FEBS Letters
Volume 582, Issue 6 , Pages 861-868, 19 March 2008

Up-regulation of heme oxygenase-1 expression through the Rac1/NADPH oxidase/ROS/p38 signaling cascade mediates the anti-inflammatory effect of 15-deoxy-Δ12,14-prostaglandin J2 in murine macrophages

Edited by Robert Barouki

  • Hye-Young Hong

      Affiliations

    • Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea
    • ,
  • Woo-Kwang Jeon

      Affiliations

    • Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea
    • ,
  • Byung-Chul Kim

      Affiliations

    • Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea
    • Research Institute of Life Sciences, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea
    • Corresponding Author InformationCorresponding author. Address: Department of Biochemistry and Research Institute of Life Sciences, College of Natural Sciences, Kangwon National University, 192-1 Hyoja-2-dong, Chuncheon 200-701, Republic of Korea. Fax: +82 33 242 0459.

Received 22 November 2007; received in revised form 6 January 2008; accepted 7 February 2008. published online 19 February 2008.

Article Outline

Abstract 

We investigated the signaling pathway that leads to the expression of heme oxygenase-1 (HO-1) in murine macrophages in response to 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2). 15dPGJ2 caused dose- and time-dependent activation of Rac1, followed by a transient increase in reactive oxygen species (ROS) via NADPH oxidase, which leads to downstream activation of p38 kinase. Inhibition of 15dPGJ2-dependent HO-1 expression significantly attenuated suppression by 15dPGJ2 of LPS-induced iNOS expression and subsequent production of nitric oxide (NO). Our findings strongly suggest that 15dPGJ2 exerts its anti-inflammatory activity through the Rac1-NADPH oxidase-ROS-p38 signaling to the up-regulation of HO-1 in an in vitro inflammation model.

Keywords: 15-Deoxy-Δ12,14-prostaglandin J2, Heme oxygenase-1, Rac1, NADPH oxidase, p38, Nitric oxide

 

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1. Introduction 

15-Deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) is an immunoregulatory lipid metabolite derived from prostaglandin D2 (PGD2) dehydration in vivo, which is abundantly produced by mast cells, dendritic cells, and alveolar macrophages [1], [2], [3]. As a ligand for the peroxisome proliferators activated receptor-γ (PPAR-γ), 15dPGJ2 exhibits anti-inflammatory effects that modulate the vascular inflammation and the atherosclerosis process [4], [5]. However, other accumulating data suggest that the PPAR-γ-independent mechanism may account for the major anti-inflammatory action of 15dPGJ2. For example, 15dPGJ2 repress several pro-inflammatory genes, including inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) genes in macrophages derived from PPAR-γ-deficient embryonic stem cells [6], [7]. 15dPGJ2 also performs its anti-inflammatory activity through direct inhibition of signaling steps leading to nuclear factor-kappa B (NF-kB) activation [8], [9], eventually down-regulating expression of pro-inflammatory cytokines and immune mediators.

Heme oxygenase-1 (HO-1) is a microsomal enzyme, catalyzing the breakdown of heme into equimolar amounts of carbon monoxide (CO), biliverdin, and free iron using molecular oxygen and reducing equivalents from NADPH:cytochrome P450 reductase [10], [11]. HO-1 contributes to the host defense reaction against oxidative injury through the antioxidant activities of biliverdin and its metabolite, bilirubin, as well as the anti-inflammatory action of CO [12], [13]. A growing body of evidence revealed that 15dPGJ2 induces HO-1 expression through mechanisms independent of PPAR-γ activation and dependent on cellular redox state in various cell types [14], [15]. Furthermore, the anti-inflammatory actions of 15dPGJ2 are functionally correlated with the induction of HO-1 expression [16], suggesting an important role of HO-1 in PPAR-γ-independent mechanism for anti-inflammatory action of 15dPGJ2. Despite these reports, the detailed signaling pathway by which 15dPGJ2 mediates induction of HO-1 expression remains largely unknown.

Regarding the signaling mechanisms of HO-1 induction, several studies have suggested involvement of reactive oxygen species (ROS) [17], [18]. ROS, such as hydrogen peroxide (H2O2), superoxide () and hydroxyl radical (OH), are important regulator of various biological processes, including cell proliferation, apoptosis, and inflammation. An important ROS source within phagocytic cells is the NADPH oxidase, which consists of two transmembrane flavocytochrome b components (gp91phox and p22phox) and the three cytosolic components (p47phox, p67phox, and p40phox), and Rac1, a member of the Rho family GTPases, plays a central role in activating this complex [19]. We here demonstrate that 15dPGJ2 exerts its in vitro anti-inflammatory activity through the Rac1-NADPH oxidase-ROS-p38 signaling to the up-regulation of HO-1.

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2. Materials and methods 

2.1. Materials 

15dPGJ2 was obtained from Biomol (Plymouth Meeting, PA). Lipopolysaccharide (LPS), diphenylenepropodiumiodide (DPI), apocynin, N-acetylcysteine (NAC), reduced glutathione (GSH) and buthionine sulfoximine (BSO) were purchased from Sigma Chemical Co. (St. Louis, MI, USA). The following were obtained from Calbiochem (La Jolla, CA): U0126, a MAPK kinase inhibitor; PD169316, a p38 inhibitor; SP600125, a JNK inhibitor. Small interfering RNAs for control, mouse p47phox and mouse p67phox were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

2.2. DNA constructs 

pEXV, pEXV-Rac1N17, pEXV-Cdc42N17, pEXV-RhoAN19, and pEXV-Rac1V12 were a kind gift from Dr. Alan Hall (Department of Biochemistry and Molecular Biology, University College London, London, UK). The plasmid pHO-1-Luc was provided by Dr. Norbert Leitinger (Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna, Austria) and contains a 4.9-kb human promoter upstream of a luciferase gene as described previously [20]. The mouse iNOS promoter-luciferase plasmid DNA, piNOS-Luc, was gift from Dr. Young-Myeong Kim (Kangwon National University, Chuncheon, Korea).

2.3. Cell culture and generation of stable cell lines 

RAW264.7, a mouse macrophage cell line, obtained from American Type Culture Collection (Manassas, VA), was cultured in Dulbecco’s modified Eagle’s medium supplemented with 2mM l-glutamine, 10% heat-inactivated fetal bovine serum, 100U/ml penicillin, and 100μg/ml streptomycin. Cells were co-transfected with pcDNA and pEXV, or pEXV-Cdc42N17, encoding a dominant negative Cdc42 mutant, or pEXV-Rac1N17, encoding a dominant negative Rac1 mutant, or pEXV-RhoAN19, encoding a dominant negative RhoA mutant, using the FuGENE 6 (Roche, Mannheim, Germany) transfection method. One day after transfection, cells were selected for G418 (Invitrogen, Carlsbad, CA) resistance. After two weeks of selection, G418-resistant clones were populated and analyzed using anti-c-myc mouse Ab (9E10; Santa Cruz Biotechnology).

2.4. DNA transfection and luciferase assay 

The RAW264.7 cells were transfected using FuGENE 6. To control for variation in transfection efficiency, all clones were co-transfected with 0.2μg of CMV-β-GAL, a eukaryotic expression vector in which Escherichia coli β-galactosidase (Lac Z) structural gene is under the transcriptional control of the CMV promoter. Cytosolic extracts were prepared 24h after transfection by using a luciferase cell lysis buffer (Promega, Madison, WI). Luciferase reporter activity was assessed on a luminometer with a luciferase assay system (Promega) according to the manufacturer’s protocol. Transfection experiments were performed in triplicate with two independently isolated sets, and the results were averaged.

2.5. Rac1 activity assay 

Rac1 activity was measured as described previously [21] using a GST-p21-activated serine/threonine protein kinase (PAK)-PAK-binding domain (PBD) fusion protein that binds GTP-bound, activated Rac1. Briefly, the fusion protein was expressed in E. coli BL21 transformed with pGEX-4T3 plasmid by incubation with isopropyl-1-thio-β-d-galactopyranoside and then purified by column chromatography using glutathione-Sepharose-4B beads. RAW264.7 cells were serum-starved (0.1% FBS) for 4h, stimulated with 1μM 15dPGJ2 for the indicated times or with the indicated concentrations of 15dPGJ2 for 5min, and then lysed in lysis buffer (150mM NaCl, 20mM Tris–Cl (pH 7.4), 2.5mM sodium pyrophosphate, 1% Triton X-100, 100mM NaF, 1mM PMSF, and 1mM Na2VO3). The lysates were centrifuged for 15min at 12000×g, after which the supernatant was incubated for 30min at 4°C with GST-(PAK)-PBD fusion protein freshly coupled to glutathione-agarose beads. The proteins that were complexed to the beads were recovered and subjected to 15% SDS–PAGE. The resolved proteins were probed with anti-Rac1 mouse Ab (BD Biosciences, San Jose, CA). The detected proteins were visualized using HRP-conjugated donkey anti-mouse IgG Ab and an ECL detection kit (Pharmacia Biotech, Uppsala, Sweden).

2.6. Immunoblot analysis 

Cytosolic extracts were obtained in 1% Triton X-100 lysis buffer (50mM Tris–Cl, pH 8.0, 150mM sodium chloride, 1mM EDTA, 1mM EGTA, 2.5mM sodium pyrophosphate, 1mM sodium orthovanadate, 1mM β-glycerophosphate, 1μg/ml leupeptin, and 1mM phenylmethylsulfonyl fluoride). Western blotting was performed using anti-HO-1 (H105; Santa Cruz Biotechnology), anti-iNOS (54; Transduction Laboratories, Lexington, KY), anti-phospho-p38 (3D7; Cell Signaling Technology, Beverly, MA), and anti-β-actin (AC-15; Sigma) antibodies. Protein samples were heated at 95°C for 5min and analyzed by SDS–PAGE. Immunoblot signals were developed by Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL).

2.7. Measurement of intracellular ROS 

For analysis of intracellular ROS, the redox-sensitive fluorescent probe DCFH-DA was used, as previously described [22]. Cells were incubated with 5μM DCFH-DA for 30min at 37°C. The harvested cells were immediately analyzed by a flow cytometry.

2.8. Indirect immunofluorescence 

RAW264.7 cells were plated at 1×105cells onto 18mm glass coverslips 24h prior to treatments. The cells were serum-starved for 4h in serum-free culture medium, and incubated with 1μM 15dPGJ2 for various time periods. The coverslips were fixed in cold 3.5% paraformaldehyde for 5min, permeabilized in cold methanol for 2min and incubated for 5min in 50mM glycine. The p67phox and Rac1 were then detected by incubation with anti-Rac1 rabbit polyclonal antibody (23A8; Upstate, VA) and anti-p47phox rabbit polyclonal antibody (H-195; Santa Cruz Biotechnology) for 2h at room temperature. After washing in phosphate-buffered saline (PBS), the coverslips were incubated for 1h at room temperature with fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and rhodamine (TRITC) conjugated goat anti-rabbit IgG secondary antibodies (Molecular Probes). Stained cells were washed, mounted in medium containing 4,6-diamino-2-phenylindole (DAPI) (h-1200; Vector Laboratories, Burlingame, CA) and examined using a Olympus Fluoview-FV300 confocal microscope.

2.9. Nitrite assay 

The nitrite level is generally accepted as indicative of NO production. Accumulated nitrite in cell culture supernatants was determined using a colorimetric assay based on the Griess reaction [23]. The supernatants (100μl) were reacted with a 100μl Griess reagent (6mg/ml) at room temperature for 10min, and then concentration was determined by measuring absorbance at 540nm. A standard curve was constructed using known concentrations of sodium nitrite.

2.10. Statistical analysis 

All data presented are expressed as means±S.D., and represent three independent experiments. Statistical analyses were assessed by unpaired Student’s t-test. Results were considered significant at P<0.05.

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3. Results and discussion 

3.1. Induction of HO-1 expression by 15dPGJ2 is dependent on Rac1 activation 

A previous study has implicated the role of reactive oxygen species (ROS) in up-regulation of HO-1 by 15dPGJ2 in human lymphocytes [14]. In phagocytes, an importance source of ROS is NADPH oxidase, and Rac1, a member of Rho family GTPases, plays a central role in activating this complex. To assess whether Rac1 is involved in the 15dPGJ2 signaling pathway leading to HO-1 gene expression, we generated cell lines stably expressing a dominant negative mutant of Cdc42, Rac1, or RhoA (Cdc42N17, Rac1N17, or RhoAN19, respectively) in RAW 264.7 cells. Treatment of RAW264.7pEXV control cells with 15dPGJ2 induced HO-1 expression (Fig. 1A, upper panel) and HO-1 promoter activity (Fig. 1A, lower panel) in a dose-dependent manner. This effect was evident at concentrations of 15dPGJ2 as low as 0.1μM. Using 1μM of 15dPGJ2 for varying time periods, induction of HO-1 expression (Fig. 1B, upper panel) and HO-1 promoter activity (Fig. 1B, lower panel) reached a maximum at 24h after addition of 15dPGJ2. Notably, both RAW264.7pEXV-Cdc42N17 and RAW264.7pEXV-RhoAN19 cells treated with 15dPGJ2 stimulated HO-1 protein expression (Fig. 1C, upper panel) and HO-1 promoter activity (Fig. 1C, lower panel) as efficiently as the control cells, whereas this stimulating effect was remarkably reduced in cells expressing Rac1N17, suggesting that Rac1 is a key mediator in 15dPGJ2 signaling to HO-1 expression. Consistent with the proposed mediatory role of Rac1 in 15dPGJ2 signaling, a significant stimulation of cellular Rac1 activity was induced, which was followed by the addition of 15dPGJ2 in time- (Fig. 1D) and dose- (Fig. 1E) dependent manner.

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  • Fig. 1. 

    Role of Rac1 in 15dPGJ2-induced HO-1 expression in murine macrophages. RAW264.7 cells were transiently transfected with HO-1 promoter-responsive luciferase reporter gene (pHO-1-Luc). The transfectants were treated with the indicated concentrations of 15dPGJ2 for 24h (A) or with 1μM 15dPGJ2 for the indicated times (B). Cell lysates were fractionated on 12% SDS–PAGE to determine the level of HO-1 and β-actin protein expression (upper panels of A and B), and the luciferase activity of HO-1 promoter was measured by luminometer as described in Section 2 (lower panels of A and B). (C) RAW264.7pEXV, RAW264.7pEXV-Cdc42N17, RAW264.7pEXV-Rac1 N17 and RAW264.7pEXV-RhoAN19 cells, which were transiently transfected with the pHO-1-Luc, incubated without or with 1μM 15dPGJ2 for 24h. HO-1 and β-actin protein levels (upper panel) and the luciferase activity of HO-1 promoter (lower panel) were analyzed as described in A and B. In low panels of A, B and C, bars depict the means±S.D. of three independent experiments. The serum-starved RAW264.7 cells were treated with 1μM 15dPGJ2 for the indicated times (D) or with the indicated concentrations for 5min (E). The cell lysates were incubated with GST-PAK-PBD that was coupled to glutathione-agarose beads. Bound Rac-GTPase was eluted and resolved by 15% SDS–PAGE. Rac-GTPase activity was detected using an anti-Rac1 antibody as a probe. As a control of protein quantity, the total amount of Rac1 was also determined by Western blotting analysis.

3.2. 15dPGJ2 induces HO-1 expression via a Rac1-NADPH oxidase-ROS pathway 

It has been known that NADPH oxidase activation requires stimulus-induced translocation of the cytosolic regulatory proteins, including p47phox and p67phox, from cytosol to the membrane-associated flavocytochrome b558 [24], and Rac1 plays a central role in mediating reactive oxygen species (ROS) production derived from NADPH oxidase in many phagocytic cells. To determine whether p47phox is involved in Rac1-dependent 15dPGJ2 signaling, we first analyzed the time-course subcellular localization of Rac1 and p47phox on 15dPGJ2 treatment. Endogenous Rac1 and p47phox detected in RAW264.7 cells were distributed throughout the cytoplasm and nucleus (Fig. 2A). However, 15dPGJ2 treatment resulted in dramatic rearrangements of the endogenous Rac1 and p47phox, leading to predominant accumulation on the plasma membrane within 5min (Fig. 2A). We next examined whether both Rac1 and NADPH oxidase are involved in 15dPGJ2-evoked ROS generation. To do this, RAW264.7pEXV or RAW264.7pEXV-Rac1 N17 cells were treated with 15dPGJ2, and changes in the intracellular levels of ROS were monitored using the H2O2-sensitive fluorescence dye DCFH-DA. As shown in Fig. 2B, 15dPGJ2-induced increases in DCF fluorescence was completely abolished in RAW264.7pEXV-Rac1N17 cells. These results suggest that 15dPGJ2 produces intracellular ROS via Rac1/NADPH oxidase in murine macrophages.

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  • Fig. 2. 

    Role of Rac1 in 15dPGJ2-induced ROS production. (A) Cells were treated with 1μM 15dPGJ2 for the indicated times. Cells were then fixed, and the immunofluorescence staining with antibodies against Rac1 (green) and p47phox (red) was performed, and visualized with confocal microscopy as described in Section 2. (B) RAW264.7pEXV and RAW264.7pEXV-Rac1N17 cells were pretreated with the H2O2-sensitive fluorescence dye DCFH-DA for 20min, and then stimulated with 1μM 15dPGJ2 for 10min. DCF fluorescence was quantified as described in Section 2.

Next, we examined any role of ROS for the up-regulation of HO-1 in response to 15dPGJ2. As shown in Fig. 3, two thiol antioxidants N-acetylcysteine (NAC) and glutathione (GSH) completely inhibited 15dPGJ2-induced HO-1 expression (upper panel) and HO-1 promoter activity (lower panel). Western blot analysis showed that transfection of siRNA specific for p47phox dose-dependently suppressed the expression of HO-1 induced by15dPGJ2 (Fig. 3B). Furthermore, Rac1V12, a constitutively active mutant of Rac1, stimulated HO-1 promoter activity, and this effect was significantly attenuated by siRNAs specific for p47phox and p67phox, two cytosolic components of NADPH oxidase (Fig. 3C). Collectively, 15dPGJ2 induces HO-1 expression in the non-stimulated RAW264.7 macrophage cells through the mediation of Rac1 followed by NADPH oxidase/ROS signaling in sequence.

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  • Fig. 3. 

    Involvement of Rac1-NADPH oxidase-ROS cascade in 15dPGJ2 – induced HO-1 expression. (A) RAW264.7 cells, which were transfected with pHO-1-Luc, were preincubated with 20μM BSO, 5mM NAC or 2mM GSH for 30min and then treated or not with 1μM 15dPGJ2 for 24h. Cell lysates were fractionated on 12% SDS–PAGE to determine the level of HO-1 and β-actin protein expression (upper panel), and the luciferase activity of HO-1 promoter was measured by luminometer as described in Section 2 (lower panel). Bars depict the means±S.D. of three independent experiments. (B) RAW264.7 cells, which were transfected with control or indicated concentrations of p47phox siRNA oligonucleotides, were incubated with1 μM 15dPGJ2 for 24h. HO-1 and β-actin protein expression levels were determined by Western blotting. (C) pEXV or pEXV-Rac1V12 was co-transfected with pHO-1-Luc and control, p47phox or p67phox siRNA oligonucleotide. Transfectants were incubated for 24h prior to luciferase assay. Bars depict the means±S.D. of three independent experiments.

3.3. p38 kinase acts downstream of ROS in 15dPGJ2 signaling to HO-1 expression 

Recent studies have shown that signaling pathways involving mitogen-activated protein kinases (MAPKs) are ultimately responsible for on HO-1 induction by stress stimuli [14], [25]. So, we tested the effects of three MAPK inhibitors on up-regulation of HO-1 expression by 15dPGJ2 in macrophages. The 15dPGJ2-mediated increase in HO-1 protein level was dose-dependently blocked by PD169316, a specific inhibitor of p38, whereas similar concentrations of U0126, a specific inhibitor of MAP/extracellular-signal regulated kinase1/2 (ERK1/2) kinase (MEK), or SP600125, a specific inhibitor of c-Jun NH2-terminal kinase1/2 (JNK1/2), had no significant effect (Fig. 4A). Consistent with this result, 15dPGJ2-induced HO-1 promoter activation was also dose-dependently abolished in cells transfected with dominant-negative mutant of p38 (DNp38) (Fig. 4B), suggesting that p38 has a specific role in 15dPGJ2 signaling to HO-1 expression. To determine whether Rac1 activation is situated up- or downstream of p38 in the 15dPGJ2 signaling pathway, we examined the phosphorylation of p38 after 15dPGJ2 stimulation in RAW264.7pEXV and RAW264.7pEXV-Rac1N17 cells. We found that 15dPGJ2 time-dependently increased phosphorylation of p38 in RAW264.7pEXV cells whereas this effect was completely blocked by stable expression of Rac1N17 (Fig. 4C). In addition, 15dPGJ2-induced transactivating potential of GAL4-ATF2 was significantly decreased by siRNAs specific for p47phox and p67phox (Fig. 4D). It thus appears that 15dPGJ2 stimulates the production of ROS via Rac1-NADPH oxidase-linked signaling cascade, this leads to p38 kinase activation and subsequently to HO-1 expression.

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  • Fig. 4. 

    Involvement of p38 as a downstream mediator of ROS in 15dPGJ2 signaling. (A) Cells were pretreated with indicated concentrations of U0126, PD169316 or SP600125 for 30min before challenge with 1μM 15dPGJ2 for 24h. HO-1 and β-actin protein levels were determined by Western blotting analysis. (B) RAW264.7 cells, which were co-transfected with pHO-1-Luc and the indicated quantities of DNp38, were treated or not with 1μM 15dPGJ2 for 24h prior to luciferase assay. Bars depict the means±S.D. of three independent experiments. (C) RAW264.7pEXV and RAW264.7pEXV-Rac1N17 cells were incubated with 1μM 15dPGJ2 for the indicated times. The extent of phosphorylation of p38 was determined by Western blotting analysis. (D) RAW264.7 cells, which were co-transfected with pFR-Luc and GAL4-ATF2, were re-transfected with control, p47phox or p67phox siRNA oligonucleotide. Transfectants were incubated with 1μM 15dPGJ2 for 24h prior to luciferase assay. Bars depict the means±S.D. of three independent experiments.

3.4. Suppression by 15dPGJ2 of LPS-stimulated NO production is mediated through the Rac1-dependent signaling to up-regulation of HO-1 

Lipopolysaccharide (LPS) is a bacterial endotoxin, which promotes the secretion of pro-inflammatory cytokines, including inducible nitric oxide synthase (iNOS) and provides an important cytotoxic function in macrophages. Pretreatment of RAW264.7 cells with 15dPGJ2 suppressed LPS-induced iNOS expression (Fig. 5, upper panel) and production of nitric oxide (NO) (Fig. 5A, lower panel) in a dose-dependent manner. The involvement of HO-1 in the anti-inflammatory action of 15dPGJ2 was tested using a specific HO competitive inhibitor, zinc protoporphyrin IX (ZnPP), or an inactive compound, copper protoporphyrin IX (CuPP). Pretreatment with ZnPP gave rise to suppression in the inhibition by 15dPGJ2 of LPS-stimulated iNOS expression (Fig. 5B, upper panel) and iNOS promoter activity (Fig. 5B, lower panel), whereas pretreatment with CuPP had no effect on the inhibition by 15dPGJ2 of LPS-stimulated iNOS expression and iNOS promoter activity, consistent with several recent reports that HO-1 negatively regulates the LPS-induced iNOS expression [26], [27]. We finally determined whether Rac1 mediates the inhibitory effect of 15dPGJ2 on LPS-induced iNOS expression and NO production. As shown in Fig. 5C, 15dPGJ2-mediated suppression of LPS-stimulated iNOS expression (upper panel) and NO production (lower panel) was significantly attenuated in RAW264.7pEXV-Rac1N17 cells compared with the RAW264.7pEXV cells, suggesting that Rac1 is involved in 15dPGJ2-mediated suppression of LPS-induced NO production in murine macrophages.

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  • Fig. 5. 

    Rac1 is required for the suppression by 15dPGJ2 of LPS-induced NO production. (A) Cells were pretreated with the indicated concentrations of 15dPGJ2 for 3h before challenge with LPS (1μg/ml) for 24h. Whole lysates were analyzed for iNOS expression by Western blotting (upper panel). The nitrites accumulation in culture medium was determined with Griess reagent (lower panel). (B) RAW264.7 cells were transfected with murine iNOS promoter. The cells were incubated without or with 1μM 15dPGJ2 for 3h in the absence or presence of ZnPP (1μM) or CuPP (1μM), followed by treatment without or with 1μg/ml LPS for additional 24h. Whole lysates were analyzed for iNOS, β-actin and HO-1 expression by Western blotting (upper panel). Luciferase activities were muasured as described in Section 2 (lower panel). Bars depict the means±S.D. of three independent experiments. (C) RAW264.7pEXV (V) and RAW264.7pEXV-Rac1 N17 (N) cells were preincubated with 1μM 15dPGJ2 for 3h prior to LPS (1μg/ml) for 24h. Whole lysates were analyzed for iNOS expression by Western blotting (upper panel). The nitrites accumulation in culture medium was determined with Griess reagent (lower panel). Bars depict the means±S.D. of three independent experiments. ∗P<0.05.

HO-1 is believed to beneficially play a cytoprotective role in a variety of pathological models such as inflammation. Carbon monoxide, a key product of HO, inhibits iNOS mRNA induction in cytokine-stimulated intestinal epithelial cells [12]. Bilirubin, a potent antioxidant, is produced from biliverdin, markedly inhibits iNOS expression and NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. However, despite its role as a potent anti-inflammatory mediator, the intracellular signaling pathway leading to the induction of HO-1 expression is not well understood. The present study provides the first evidence of the pivotal roles played by Rac1 and NADPH oxidase in the anti-inflammatory signaling by which 15dPGJ2 induces the expression of HO-1 in RAW264.7 macrophages.

LPS is a bacterial endotoxin, which promotes the secretion of pro-inflammatory cytokines and related molecules, including iNOS in many cell types. Lanone et al. [28] reported that NADPH oxidase activity is required for LPS-induced iNOS expression in RAW264.7 macrophages, and this finding is opposite to the role of NADPH oxidase showed in the present study. Many studies, however, relied on the results obtained with DPI or apocyanin at micromolar or millimolar concentrations. Besides acting as an NADPH oxidase-like flavoenzyme inhibitor, DPI has been also reported to be a potent inhibitor of iNOS with an IC50 of 50nM [29]. In line with this, Heumüller et al. [30] reported that apocynin is not an inhibitor of vascular NADPH oxidase. We also observed that DPI at submicromolar concentrations completely blocked LPS-induced NO production, whereas DPI at the same condition had a little effect to inhibits the LPS-induced iNOS expression (see Fig. 6A, which is published in supporting information). In addition, LPS-induced iNOS expression and NO production were little affected by two thiol antioxidants NAC and GSH, and exogenous hydrogen peroxide failed to induces iNOS expression and subsequent NO production (see Fig. 6B, C and D, which is published in supporting information). From these findings, we carefully imply that ROS is a minor contributor in LPS-evoked intracellular signaling to inflammatory response, and the major effect of DPI on suppression of LPS-induced NO production is primarily due to the inhibition of iNOS activity rather than the inhibition of NADPH oxidase. Furthermore, support for this conception comes from the observation that p47phox siRNA transfection inhibited the induction of anti-inflammatory enzyme, HO-1 in response to 15dPGJ2.

In conclusion, our data provide, for the first time, evidence that 15dPGJ2 induces HO-1 expression in murine RAW264.7 macrophages, which is mediated via Rac1/NADPH oxidase-dependent generation of ROS leading to the activation of p38 mitogen-activated protein (MAP) kinase. Furthermore, our results suggest that HO-1 up-regulation via the Rac1/NADPH oxidase/ROS/p38 cascade is critical for the anti-inflammatory action of 15dPGJ2.

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Acknowledgement 

This work was supported by a Grant (No. R01-2003-000-10029-0) from the Basic Research Program of the Korea Science and Engineering Foundation, Korea.

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Appendix A. Supplementary data 

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  • Fig. 6. 

    Effects of DPI, NAC, GSH and H2O2 on LPS-induced iNOS expression and NO production. Cells were preincubated with the indicated concentrations of DPI (A), GSH (B), NAC (C) or H2O2 (D) before challenge with 1 μg/ml LPS (A, B and C) for additional 24 h. Whole lysates were analyzed for iNOS expression by Western blotting (upper panels of A, B,C and D). The nitrites accumulation in culture medium was determined with Griess reagent (lower panels of A, B, C and D).

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PII: S0014-5793(08)00117-8

doi:10.1016/j.febslet.2008.02.012

FEBS Letters
Volume 582, Issue 6 , Pages 861-868, 19 March 2008

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