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• W2096073982 abstract "15-Deoxy-Δ12,14-prostaglandin J2 (15dPGJ2 has been recently proposed as a potent anti-inflammatory agent. However, the mechanisms by which 15dPGJ2 mediates its therapeutic effects in vivo are unclear. We demonstrate that 15dPGJ2 at micromolar (2.5–10 μm) concentrations induces the expression of heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, at both mRNA and protein levels in human lymphocytes. In contrast, troglitazone and ciglitazone, two thiazolidinediones that mimic several effects of 15dPGJ2 through their binding to the peroxisome proliferator-activated receptor (PPAR)-γ, did not affect HO-1 expression, and the positive effect of 15dPGJ2 on this process was mimicked instead by other cyclopentenone prostaglandins (PG), such as PGD2 (the precursor of 15dPGJ2) and PGA1 and PGA2 which do not interact with PPAR-γ. Also, 15dPGJ2 enhanced the intracellular production of reactive oxygen species (ROS) and increased xanthine oxidase activity in vitro. Inhibition of intracellular ROS production by N-acetylcysteine, TEMPO, Me2SO, 1,10-phenanthroline, or allopurinol resulted in a decreased 15dPGJ2-dependent HO-1 expression in the cells. Furthermore, buthionine sulfoximine, an inhibitor of reduced glutathione synthesis, or Fe2+/Cu2+ ions enhanced the positive effect of 15dPGJ2 on HO-1 expression. On the other hand, the inhibition of phosphatidylinositol 3-kinase or p38 mitogen-activated protein kinase, or the blockade of transcription factor NF-κB activation, hindered 15dPGJ2-elicited HO-1 expression. Collectively, the present data suggest that 15dPGJ2 anti-inflammatory actions at pharmacological concentrations involve the induction of HO-1 gene expression through mechanisms independent of PPAR-γ activation and dependent on ROS produced via the xanthine/xanthine oxidase system and/or through Fenton reactions. Both phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase signaling pathways also appear implicated in modulation of HO-1 expression by 15dPGJ2. 15-Deoxy-Δ12,14-prostaglandin J2 (15dPGJ2 has been recently proposed as a potent anti-inflammatory agent. However, the mechanisms by which 15dPGJ2 mediates its therapeutic effects in vivo are unclear. We demonstrate that 15dPGJ2 at micromolar (2.5–10 μm) concentrations induces the expression of heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, at both mRNA and protein levels in human lymphocytes. In contrast, troglitazone and ciglitazone, two thiazolidinediones that mimic several effects of 15dPGJ2 through their binding to the peroxisome proliferator-activated receptor (PPAR)-γ, did not affect HO-1 expression, and the positive effect of 15dPGJ2 on this process was mimicked instead by other cyclopentenone prostaglandins (PG), such as PGD2 (the precursor of 15dPGJ2) and PGA1 and PGA2 which do not interact with PPAR-γ. Also, 15dPGJ2 enhanced the intracellular production of reactive oxygen species (ROS) and increased xanthine oxidase activity in vitro. Inhibition of intracellular ROS production by N-acetylcysteine, TEMPO, Me2SO, 1,10-phenanthroline, or allopurinol resulted in a decreased 15dPGJ2-dependent HO-1 expression in the cells. Furthermore, buthionine sulfoximine, an inhibitor of reduced glutathione synthesis, or Fe2+/Cu2+ ions enhanced the positive effect of 15dPGJ2 on HO-1 expression. On the other hand, the inhibition of phosphatidylinositol 3-kinase or p38 mitogen-activated protein kinase, or the blockade of transcription factor NF-κB activation, hindered 15dPGJ2-elicited HO-1 expression. Collectively, the present data suggest that 15dPGJ2 anti-inflammatory actions at pharmacological concentrations involve the induction of HO-1 gene expression through mechanisms independent of PPAR-γ activation and dependent on ROS produced via the xanthine/xanthine oxidase system and/or through Fenton reactions. Both phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase signaling pathways also appear implicated in modulation of HO-1 expression by 15dPGJ2. 15-Deoxy-Δ12,14-prostaglandin J2 (herein referred to as 15dPGJ2) 1The abbreviations used are: 15dPGJ2, 15-deoxy-Δ12,14-prostaglandin J2; PPAR, peroxisome proliferator-activated receptor; MAP, mitogen-activated protein; ROS, reactive oxygen species; HO-1, heme oxygenase-1; NAC, N-acetylcysteine; BSO, buthionine sulfoximine; HMAP, 4-hydroxy-3-methoxyacetophenone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DCFDA, 2,7-dichlorohydrofluorescein diacetate; O2⋅¯, superoxide anion; ·OH, hydroxyl radicals; ERK1/2, extracellular signal-regulated kinases 1 and 2; PI, phosphatidylinositol; RT, reverse transcriptase. 1The abbreviations used are: 15dPGJ2, 15-deoxy-Δ12,14-prostaglandin J2; PPAR, peroxisome proliferator-activated receptor; MAP, mitogen-activated protein; ROS, reactive oxygen species; HO-1, heme oxygenase-1; NAC, N-acetylcysteine; BSO, buthionine sulfoximine; HMAP, 4-hydroxy-3-methoxyacetophenone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DCFDA, 2,7-dichlorohydrofluorescein diacetate; O2⋅¯, superoxide anion; ·OH, hydroxyl radicals; ERK1/2, extracellular signal-regulated kinases 1 and 2; PI, phosphatidylinositol; RT, reverse transcriptase. is an active metabolite derived from PGD2 dehydration in vivo, which is physiologically present in body fluids at 10–12 to 10–9 m concentrations. However, 15dPGJ2 amounts become highly increased in several pathological conditions, such as infection and inflammation (1Straus D.S. Glass C.K. Med. Res. Rev. 2001; 21: 185-210Crossref PubMed Scopus (544) Google Scholar, 2Gilroy D.W. Colville-Nash P.R. Willis D. Chivers J. Paul-Clark M.J. Willoughby D.A. Nat. Med. 1999; 5: 698-701Crossref PubMed Scopus (1112) Google Scholar). PGD2 and thus 15dPGJ2 are abundantly produced by mast cells, platelets, and alveolar macrophages, and 15dPGJ2 has been proposed as a key immunoregulatory lipid mediator (3Ito S. Narumiya S. Hayaishi O. Prostaglandins Leukotrienes Essent. Fatty Acids. 1989; 37: 219-234Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 4Kawahito Y. Kondo M. Tsubouchi Y. Hashiramoto A. Bishop-Bailey D. Inoue K. Kohno M. Yamada R. Hla T. Sano H. J. Clin. Investig. 2000; 106: 189-197Crossref PubMed Scopus (366) Google Scholar, 5Su C.G. Wen X. Bailey S.T. Jiang W. Rangwala S.M. Keilbaugh S.A. Flanigan A. Murthy S. Lazar M.A. Wu G.D. J. Clin. Investig. 1999; 104: 383-389Crossref PubMed Scopus (720) Google Scholar). A number of its actions seem to be exerted through its binding to peroxisome proliferator-activated receptor (PPAR)-γ (6Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1859) Google Scholar, 7Harris S.G. Phipps R.P. Eur. J. Immunol. 2001; 31: 1098-1105Crossref PubMed Scopus (175) Google Scholar, 8Clark R.B. Bishop-Bailey D. Estrada-Hernandez T. Hla T. Puddington L. Padula S.J. J. Immunol. 2000; 164: 1364-1371Crossref PubMed Scopus (438) Google Scholar). The PPARs are nuclear hormone receptors that can bind, normally as heterodimers with the retinoid X receptor, to peroxisome proliferator-responsive elements (AGGTCAnAGGTCA) present in the promoters of their target genes to activate their transcription (9Yu K. Bayona W. Kallen C.B. Harding H.P. Ravera C.P. McMahon G. Brown M. Lazar M.A. J. Biol. Chem. 1995; 270: 23975-23983Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). A variety of immune cells, including T and B lymphocytes as well as macrophages, have been found to express PPAR-γ and their activation to be regulated via PPAR-γ-dependent mechanisms (7Harris S.G. Phipps R.P. Eur. J. Immunol. 2001; 31: 1098-1105Crossref PubMed Scopus (175) Google Scholar, 8Clark R.B. Bishop-Bailey D. Estrada-Hernandez T. Hla T. Puddington L. Padula S.J. J. Immunol. 2000; 164: 1364-1371Crossref PubMed Scopus (438) Google Scholar, 10Wang Y.L. Frauwirth K.A. Rangwala S.M. Lazar M.A. Thompson C.B. J. Biol. Chem. 2002; 277: 31781-31788Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). However, it has been also demonstrated that other effects of 15dPGJ2 are exerted independently of PPAR-γ (reviewed in Ref. 1Straus D.S. Glass C.K. Med. Res. Rev. 2001; 21: 185-210Crossref PubMed Scopus (544) Google Scholar), such as the activation of mitogen-activated protein (MAP) kinase cascades in astrocytes and preadipocytes, which is mediated by reactive oxygen species (ROS) (11Lennon A.M. Ramauge M. Dessouroux A. Pierre M. J. Biol. Chem. 2002; 277: 29681-29685Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), the induction of caspase-dependent apoptosis in eosinophils and neutrophils (12Ward C. Dransfield I. Murray J. Farrow S.N. Haslett C. Rossi A.G. J. Immunol. 2002; 168: 6232-6243Crossref PubMed Scopus (120) Google Scholar), the inhibition of cell adhesion and the oxidative burst in neutrophils (13Vaidya S. Somers E.P. Wright S.D. Detmers P.A. Bansal V.S. J. Immunol. 1999; 163: 6187-6192PubMed Google Scholar), and the induction of the expression of proinflammatory genes in monocytes/macrophages derived from PPARγ-deficient embryonic stem cells (14Moore K.J. Rosen E.D. Fitzgerald M.L. Randow F. Andersson L.P. Altshuler D. Milstone D.S. Mortensen R.M. Spiegelman B.M. Freeman M.W. Nat. Med. 2001; 7: 41-47Crossref PubMed Scopus (444) Google Scholar, 15Chawla A. Barak Y. Nagy L. Liao D. Tontonoz P. Evans R.M. Nat. Med. 2001; 7: 48-52Crossref PubMed Scopus (957) Google Scholar).The role of 15dPGJ2 in the modulation of inflammation is nevertheless complex, because this prostaglandin exhibits both anti-inflammatory and proinflammatory functions and thus remains the subject of intensive investigation. In this context, 15dPGJ2 has been shown to inhibit the expression of inducible NO synthase, as well as tumor necrosis factor-α and interleukin-1β production in mouse and human macrophages, thus suggesting a role for 15dPGJ2 in the inhibition of inflammation (16Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. Nature. 1998; 391: 79-82Crossref PubMed Scopus (3241) Google Scholar, 17Jiang C. Ting A.T. Seed B. Nature. 1998; 391: 82-86Crossref PubMed Scopus (537) Google Scholar). Available data also suggest that the induction of heme oxygenase-1 (HO-1) expression elicited by Δ12-PGJ2 in rat basophilic leukemia cells and other cell types involved in the inflammatory response may play an important role in the fate of heme liberated during inflammation, and thus an anti-inflammatory role has been ascribed to Δ12-PGJ2 (18Koizumi T. Odani N. Okuyama T. Ichikawa A. Negishi M. J. Biol. Chem. 1995; 270: 21779-21784Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). In this view, 15dPGJ2 has been suggested to play a feedback regulatory role in the resolution phase of inflammation and hence is considered as a potential therapeutic target for the treatment of inflammatory diseases (1Straus D.S. Glass C.K. Med. Res. Rev. 2001; 21: 185-210Crossref PubMed Scopus (544) Google Scholar). However, caution must be taken in assigning a solely anti-inflammatory role to 15dPGJ2, because there is also experimental evidence that 15dPGJ2 can induce the synthesis of the type II-secreted proinflammatory mediators phospholipase A (6Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1859) Google Scholar) and COX-2 in smooth muscle (20Meade E.A. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1999; 274: 8328-8334Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar) and epithelial cells (21Couturier C. Brouillet A. Couriaud C. Koumanov K. Bereziat G. Andreani M. J. Biol. Chem. 1999; 274: 23085-23093Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Moreover, it has been shown recently (22Harris S.G. Smith R.S. Phipps R.P. J. Immunol. 2002; 168: 1372-1379Crossref PubMed Scopus (72) Google Scholar) that in stimulated human T lymphocytes, 15dPGJ2 induces a significant increase in interleukin-8 production through the activation of a MAP kinase- and NF-κB-dependent signaling pathway.Heme oxygenase is a widely distributed enzyme in mammalian tissues whose main function is associated with the degradation of heme to biliverdin, iron, and carbon monoxide (23Maines M.D. Trakshel G.M. Kutty R.K. J. Biol. Chem. 1986; 261: 411-419Abstract Full Text PDF PubMed Google Scholar). Three distinct isoforms of this protein have been characterized, two of which (HO-2 and HO-3) are constitutively expressed, whereas the third (HO-1) displays inducible expression (24McCoubrey Jr., W.K. Huang T.J. Maines M.D. Eur. J. Biochem. 1997; 247: 725-732Crossref PubMed Scopus (734) Google Scholar). Several pathological states, including hypoxia, atherosclerosis, and inflammation, have been found to be accompanied by overexpression of the HO-1 gene and increased HO-1 enzyme activity (25Motterlini R. Foresti R. Bassi R. Calabrese V. Clark J.E. Green C.J. J. Biol. Chem. 2000; 275: 13613-13620Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 26Ishikawa K. Navab M. Leitinger N. Fogelman A.M. Lusis A.J. J. Clin. Investig. 1997; 100: 1209-1216Crossref PubMed Scopus (255) Google Scholar, 27Willis D. Moore A.R. Frederick R. Willoughby D.A. Nat. Med. 1996; 2: 87-90Crossref PubMed Scopus (705) Google Scholar). Over the past few years, several studies (28Morse D. Pischke S.E. Zhou Z. Davis R.J. Flavell R.A. Loop T. Otterbein S.L. Otterbein L.E. Choi A.M. J. Biol. Chem. 2003; 278: 36993-36998Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 29Otterbein L.E. Choi A.M. Am. J. Physiol. 2000; 279: L1029-L1037Crossref PubMed Google Scholar) have revealed the important function of HO-1 as a cytoprotective mechanism against oxidative insults, derived from the antioxidant activities of biliverdin and bilirubin and from the anti-inflammatory action of carbon monoxide. In this context, a common denominator characterizing the prompt stimulation of HO-1 expression occurring under the above conditions is a drastic change in the intracellular redox status accompanied by a transient decrease in cytosolic reduced glutathione levels (25Motterlini R. Foresti R. Bassi R. Calabrese V. Clark J.E. Green C.J. J. Biol. Chem. 2000; 275: 13613-13620Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 30Lautier D. Luscher P. Tyrrell R.M. Carcinogenesis. 1992; 13: 227-232Crossref PubMed Scopus (180) Google Scholar).Recent studies have illustrated that 15dPGJ2 induces HO-1 expression in a variety of cells, including hepatocytes (31Gong P. Stewart D. Hu B. Li N. Cook J. Nel A. Alam J. Antioxid. Redox Signal. 2002; 4: 249-257Crossref PubMed Scopus (128) Google Scholar), cardiac myocytes (32Wayman N.S. Hattori Y. McDonald M.C. Mota-Filipe H. Cuzzocrea S. Pisano B. Chatterjee P.K. Thiemermann C. FASEB J. 2002; 16: 1027-1040Crossref PubMed Scopus (364) Google Scholar), microglial cells (33Koppal T. Petrova T.V. Van Eldik L.J. Brain Res. 2000; 867: 115-121Crossref PubMed Scopus (51) Google Scholar), murine macrophages (34Lee T.S. Tsai H.L. Chau L.Y. J. Biol. Chem. 2003; 278: 19325-19330Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), and rat basophilic leukemia cells (18Koizumi T. Odani N. Okuyama T. Ichikawa A. Negishi M. J. Biol. Chem. 1995; 270: 21779-21784Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Also, it has been shown that 15dPGJ2 is able to induce apoptosis in mouse T cells and to inhibit T cell activation (7Harris S.G. Phipps R.P. Eur. J. Immunol. 2001; 31: 1098-1105Crossref PubMed Scopus (175) Google Scholar, 8Clark R.B. Bishop-Bailey D. Estrada-Hernandez T. Hla T. Puddington L. Padula S.J. J. Immunol. 2000; 164: 1364-1371Crossref PubMed Scopus (438) Google Scholar, 35Yang X.Y. Wang L.H. Chen T. Hodge D.R. Resau J.H. DaSilva L. Farrar W.L. J. Biol. Chem. 2000; 275: 4541-4544Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). However, the effect of 15dPGJ2 on HO-1 gene expression has not yet been analyzed in human lymphocytes, and in general there is no information available on the signaling mechanisms modulating HO-1 synthesis in these cells. In the present study, we have investigated the effect of 15dPGJ2, using pharmacological concentrations, on the expression of HO-1 in human lymphocytes, and the involvement of ROS-dependent specific pathways in this process. We present clear evidence that 15dPGJ2 at micromolar (2.5–10 μm) levels induces an increase of HO-1 mRNA and protein levels in human lymphocytes, a process that is prevented by scavengers of ROS, and we postulate that ROS derived from both Fenton chemistry and the xanthine/xanthine oxidase system are implicated in the induction of HO-1 gene expression in this cell type. In addition, we provide evidence of phosphatidylinositol 3-kinase (PI3-kinase) and p38 MAP kinase acting as mediators of 15dPGJ2 effects on HO-1 expression and of the involvement of the transcription factor NF-κBin this process. These results contribute to shed some light on the mechanisms whereby 15dPGJ2 modulates leukocyte functions through PPAR-γ-independent pathways.EXPERIMENTAL PROCEDURESMaterials—15dPGJ2, SN50 (AAVALLPAVLLALLAPVQRKRQKLMP), and SN50M (AAVALLPAVLLALLAPVQRNGQKLMP) peptides were obtained from Biomol (Plymouth Meeting, PA). Troglitazone, ciglitazone, Wy14,643, PGD2, PGA1, and PGA2 were products of Cayman Chemical (Ann Arbor, MI). Sodium arsenite, actinomycin D, cycloheximide, N-acetylcysteine (NAC), buthionine sulfoximine (BSO), TEMPO, Me2SO, 1,10-phenanthroline, allopurinol, 4-hydroxy-3-methoxyacetophenone (HMAP), probenecid, luminol, lucigenin, ketoconazole, rotenone, genistein, staurosporine, cyclosporin A, okadaic acid, wortmannin, and LY294002 were purchased from Sigma. SB203580 and PD098059 were products of Calbiochem, and RPMI 1640 medium was obtained from Biomedia (Boussens, France).Isolation and Culture of Human Lymphocytes—Human peripheral blood lymphocytes were isolated from fresh heparinized blood of healthy human donors, after informed consent, by Ficoll-Paque (Amersham Biosciences) gradient centrifugation followed by hypotonic lysis of residual erythrocytes (36Carballo M. Conde M. El Bekay R. Martin-Nieto J. Camacho M.J. Monteseirin J. Conde J. Bedoya F.J. Sobrino F. J. Biol. Chem. 1999; 274: 17580-17586Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). After elimination of adherent cells by incubation on plastic dishes for 45 min at 37 °C, lymphocytes were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin and maintained at 37 °C in an atmosphere of 5% CO2. In all control experiments in which 15dPGJ2 was omitted, its vehicle (0.1% Me2SO) was added instead.Western Blotting Analysis of HO-1 Protein Levels—Cells were rinsed once with ice-cold phosphate-buffered saline, resuspended in a lysis solution containing 50 mm Tris-HCl (pH 7.4), 10 mm EDTA, 50 mm NaF, 10% glycerol, 1% Triton X-100, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride, and maintained on ice for 30 min. Then the cells were disrupted by sonication on ice and, after centrifugation at 12,000 × g for 5 min at 4 °C, the protein concentration in the supernatant was determined by the Bradford method (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar), using bovine serum albumin as a standard. Cell lysate proteins (38Naughton P. Foresti R. Bains S.K. Hoque M. Green C.J. Motterlini R. J. Biol. Chem. 2002; 277: 40666-40674Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) were boiled in Laemmli loading buffer, resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Pall, Madrid, Spain). Blots were probed with rabbit polyclonal antibodies to HO-1 (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution, and horseradish peroxidase-conjugated secondary antibodies to rabbit IgG (Sigma) were used at a 1:5000 dilution to detect the 32-kDa HO-1 band by subsequent enhanced chemiluminescence (39Carballo M. Marquez G. Conde M. Martin-Nieto J. Monteseirin J. Conde J. Pintado E. Sobrino F. J. Biol. Chem. 1999; 274: 93-100Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). To verify even protein loading, the blots were subsequently stripped and reprobed with rabbit polyclonal antibodies against β-actin (42 kDa) from Santa Cruz Biotechnology (1:1000 dilution). Relative protein levels were determined by scanning densitometry analysis using the Scion Image software. Values given below each panel in Figs. 1, 2, 3, 4 and 6, 7, 8, 9 represent the mean (in relative units), normalized to control values, of at least three experiments performed with similar results.Fig. 2Effect of synthetic PPAR agonists and cyclopentenone prostaglandins on HO-1 expression in human lymphocytes. Cells were incubated at 37 °C in the absence or presence of 5 μm 15dPGJ2, 25 μm troglitazone, 40 μm ciglitazone, 25 μm Wy14,643, 5 μm sodium arsenite (AsO2Na), 10 μm PGD2, 15 μm PGA1 or 25 μm PGA2 for 18 (A and C) or 4 h (B and D). The cells were then lysed, and HO-1 and β-actin protein levels were determined by Western blotting analysis (A and C), and HO-1 and GAPDH mRNA levels were analyzed by RT-PCR (B and D).View Large Image Figure ViewerDownload (PPT)Fig. 3Effect of NAC and BSO on 15dPGJ2-dependent HO-1 expression in human lymphocytes. Cells were preincubated at 37 °C for 1 h with the indicated doses of NAC for 1 h and then treated or not with 5 μm 15dPGJ2 for 18 h (A) or preincubated with 25 μm BSO for 1 h, and then treated with the indicated doses of 15dPGJ2 for 18 h (B). Thereafter, the cells were lysed, and HO-1 and β-actin protein levels were determined by Western blotting analysis.View Large Image Figure ViewerDownload (PPT)Fig. 4Effect of ROS scavengers on 15dPGJ2-dependent HO-1 expression in human lymphocytes. Cells were preincubated at 37 °C in the absence or presence of 100 μm TEMPO or 0.5% Me2SO for 2 h and then treated or not with 3 μm 15dPGJ2 for 18 h. Thereafter, the the cells were lysed, and HO-1 and β-actin protein levels were determined by Western blotting analysis.View Large Image Figure ViewerDownload (PPT)Fig. 6Effect of Fe2+/Cu2+ ions on 15dPGJ2-dependent HO-1 expression in human lymphocytes. Cells were preincubated at 37 °C in the absence or presence of 20 μm 1,10-phenanthroline (1,10-Phe) or FeSO4 plus CuSO4 at the indicated doses for 1 h, and then treated or not with 3 μm 15dPGJ2 for 18 h. Thereafter, the cells were lysed, and HO-1 and β-actin protein levels were determined by Western blotting analysis.View Large Image Figure ViewerDownload (PPT)Fig. 7Effects of allopurinol on 15dPGJ2-dependent HO-1 expression in human lymphocytes and of 15dPGJ2 on xanthine oxidase activity.A, cells were preincubated at 37 °C with the indicated doses of allopurinol for 1 h and then treated or not with 5 μm 15dPGJ2 for 18 h. Thereafter, the cells were lysed, and HO-1 and β-actin protein levels were determined by Western blotting analysis. B, xanthine oxidase at the indicated doses was incubated at 37 °C with vehicle (0.1% Me2SO) or 10 μm 15dPGJ2 for 20 min. Uric acid production was then measured on the basis of the absorbance at 293 nm. Plotted values are the mean ± S.E. from three separate experiments.View Large Image Figure ViewerDownload (PPT)Fig. 8Effect of PI3-kinase and MAP kinase inhibitors on 15dPGJ2-dependent HO-1 expression in human lymphocytes. Cells were preincubated at 37 °C for 30 min in the absence or presence of 100 nm wortmannin, 20 μm LY294002, 5 μm SB203580, or 25 μm PD098059 and then treated or not with 10 μm 15dPGJ2 for 4 h (A) or 5 μm 15dPGJ2 for 4 h (C), or else preincubated with the indicated doses of SB203580 or PD098059 for 2 h and then treated or not with 5 μm 15dPGJ2 for 18 h (B). Thereafter, the cells were lysed, and HO-1 and β-actin protein levels were determined by Western blotting analysis (A and B), and HO-1 and GAPDH mRNA levels were analyzed by RT-PCR (C).View Large Image Figure ViewerDownload (PPT)Fig. 9Effect of NF-κB-blocking peptide SN50 on 15dPGJ2-dependent HO-1 expression in human lymphocytes. Cells were preincubated at 37 °C in the absence or presence of 13.5 μm SN50 or SN50M peptides for 2 h, and then treated or not with 5 μm 15dPGJ2 for 18 h. Thereafter, the cells were lysed, and HO-1 and β-actin protein levels were determined by Western blotting analysis.View Large Image Figure ViewerDownload (PPT)RT-PCR Analysis of HO-1 mRNA Levels—Total cellular RNA was extracted using the phenol-guanidinium isothiocyanate method (40Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62983) Google Scholar). Reverse transcription to cDNA was performed at 37 °C for 1 h in 24 μl of reaction mixture containing 2 μg of RNA, the four dNTPs at 1 mm each, 2.5 μm random hexamer primers, 1 mm dithiothreitol, 20 units of RNAsin ribonuclease inhibitor, and 100 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). PCR amplification of cDNA (4Kawahito Y. Kondo M. Tsubouchi Y. Hashiramoto A. Bishop-Bailey D. Inoue K. Kohno M. Yamada R. Hla T. Sano H. J. Clin. Investig. 2000; 106: 189-197Crossref PubMed Scopus (366) Google Scholar) was performed in 10 μl of reaction using the following HO-1-specific primers at 0.5 μm each: forward, 5′-TTCTTCACCTTCCCCAAC-3′, and reverse, 5′-GCATAAAGCCCTACAGCAAC-3′), the four dNTPs at 400 μm each, 5% Me2SO, and 1 unit of TaqDNA polymerase (Roche Diagnostics). After an initial denaturation step at 94.5 °C for 5 min, PCR was performed for a total of 45 cycles, each at 94.5 °C for 1 min, 60 °C for 2 min, and 72 °C for 3 min. To ensure that equal amounts of cDNA were added to the PCRs, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was amplified as a control, using the following primers: forward, 5′-CCACCCATGGCAAATTCCATGGC-3′, and reverse, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′. PCR-amplified bands (HO-1, 365 bp; GADPH, 600 bp) were visualized on 1% agarose gels stained with ethidium bromide using the BioDoc-It™ system (Ultraviolet Products, Upland, CA).ROS Production Assay—2,7-Dichlorohydrofluorescein diacetate (DCFDA, from Sigma) was used as an indicator of the amount of intracellular ROS (41Cathcart R. Schwiers E. Ames B.N. Anal. Biochem. 1983; 134: 111-116Crossref PubMed Scopus (723) Google Scholar). Briefly, 5 × 106 human lymphocytes were suspended in 1 ml of KR-Hepes and incubated in the presence of 2.5 μm DCFDA and 2.5 mm probenecid (an inhibitor of anion membrane transport) at 37 °C for 1 h in the dark. The cells were then rinsed twice with KR-Hepes plus 2.5 mm probenecid, and after addition of the stimulus, fluorescence intensity was measured for up to 4 h in a Wallac 1420 VICTOR2TM spectrofluorometer, using excitation and emission wavelengths of 485 and 535 nm, respectively. In addition, luminol- and lucigenin-enhanced chemiluminescence assays were performed as described (42El Bekay R. Alvarez M. Monteseirin J. Alba G. Chacon P. Vega A. Martin-Nieto J. Jimenez J. Pintado E. Bedoya F.J. Sobrino F. Blood. 2003; 102: 662-671Crossref PubMed Scopus (151) Google Scholar) for the measurement of total ROS and superoxide anion (O2⋅¯) levels, respectively.Xanthine Oxidase Activity Assay—Xanthine oxidase activity was determined by monitoring the rate of uric acid formation (43Sau A.K. Mondal M.S. Mitra S. Biochim. Biophys. Acta. 2001; 1544: 89-95Crossref PubMed Scopus (13) Google Scholar). Assays were performed using different concentrations of cow milk xanthine oxidase (Roche Diagnostics) in a reaction mixture (1 ml) containing 0.1 mm xanthine and 0.1 mm EDTA in 50 mm phosphate potassium buffer (pH 7.4). The reactions were carried out at 37 °C and stopped at 20 min by the addition of 100 μl of 100% (w/v) trichloroacetic acid. After centrifugation at 10,000 × g for 15 min, the uric acid produced was measured in the supernatants on the basis of the change in the absorbance at 293 nm.RESULTS15dPGJ2 Induces HO-1 Gene Expression in Human Lymphocytes—Preliminary experiments were addressed to analyze the effect of 15dPGJ2 on the expression of HO-1 in human lymphocytes. Fig. 1A shows that 10 μm 15dPGJ2 elicited the synthesis of HO-1 protein in a time-dependent manner. This effect was noticeable at 3 h after 15dPGJ2 addition, reaching its maximal expression at 12 h of treatment and maintaining at this level during the rest of the experiment (up to 30 h). The induction of HO-1 protein expression correlated with increased levels of HO-1 mRNA, which became detectable as early as 1 h after treatment with 15dPGJ2 (Fig. 1B). A dose-dependent analysis showed that HO-1 gene expression was induced at both protein (Fig. 1C) and mRNA (Fig. 1D) levels by concentrations of 15dPGJ2 in the 2.5–10 μm range. In order to study the molecular mechanisms implicated in 15dPGJ2-dependent HO-1 induction, human lymphocytes were treated with actinomycin D, a well known inhibitor of transcription, and cycloheximide, an established inhibitor of protein synthesis, before the addition of 15dPGJ2 (Fig. 1, E and F). As expected, the pretreatment of lymphocytes with actinomycin D blocked both HO-1 protein and mRNA synthesis, whereas cycloheximide inhibited HO-1 protein expression but did not affect mRNA expression, suggesting th" @default.
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• W2096073982 title "15-Deoxy-Δ12,14-prostaglandin J2 Induces Heme Oxygenase-1 Gene Expression in a Reactive Oxygen Species-dependent Manner in Human Lymphocytes" @default.
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