Nrf2 德国Greifswald大学

Original Contribution PGD2 and PGE2 regulate gene expression o Saskia F. Erttmann a,b,1, Antje Bast a,⁎,1, Julia Seidel b, Ka a Friedrich Loeffler Institute of Medical Microbiology, Ernst Moritz Arndt University of Greifswa b Department of Medical Biochemistry and Molecular Biology, Ernst Moritz Arndt University o s a A2 S an ls. T essi vid ei ex eal Using stimulated macrophages from Nrf2-deficient mice or activators of Nrf2 and identified Nrf2 as a critical player mediating trans ely dis e hydr perox steine teine P sequen ianmem ic Ca2+ 2 oxidas Free Radical Biology & Medicine xxx (2011) xxx–xxx FRB-10675; No. of pages: 15; 4C: Contents lists available at ScienceDirect Free Radical Biolo j ourna l homepage: www.e lsev ie metabolism of phospholipids, such as those of lung surfactant. Prx 6 is Three kinds of enzymes, phospholipase A2, COX, and terminal PG synthases, are involved in the biosynthesis of the conventional prostaglandins PGD2 and PGE2. Cytosolic phospholipase A2 (cPLA2) catalyzes the hydrolysis of the sn-2 acyl bond of membrane phospholipids, resulting in production of lysophospholipids and release of free arachidonic acid, which is then supplied to either of the two COX isoenzymes, constitutive COX-1 (PGH synthase 1) or inducible COX-2 (PGH synthase 2). The COX metabolite PGH2 is subsequently isomerized to PGD2 or PGE2 by terminal PG synthase Abbreviations: AACOCF3, arachidonyl trifluoromethyl ketone; AC, adenylate cyclase; AGC, cAMP-dependent protein kinase A/protein kinase G/protein kinase C; BMM, bone marrow-derived macrophages; CAPE, caffeic acid phenethyl ester; COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; CREB, cAMP response element-binding protein; Epac, exchange protein directly activated by cAMP; IBMX, 3-isobutyl-1-methylxan- thine; IFN-γ, interferon-γ; iNOS, induciblenitric oxide synthase; JAK2, Januskinase 2;Keap-1, Kelch-like ECH-associated protein 1; L-NIL, L-N6-(1-iminoethyl)lysine dihydrochloride; LPS, lipopolysaccharide; MAFP, methyl arachidonyl fluorophosp ed protein kinase; Nrf2, nuclear erythroid-derived 2p45-rel PKA, protein kinase A; PPAR, peroxisomeproliferator-activa RPLP0, ribosomal protein large P0; sulforaphane, 1-iso butane; tBHQ, tert-butylhydroquinone. ⁎ Corresponding author. Fax: +49 3834 865561. E-mail address: antje.bast@uni-greifswald.de (A. Bas 1 These authors contributed equally to this work. 0891-5849/$ – see front matter © 2011 Elsevier Inc. Al doi:10.1016/j.freeradbiomed.2011.05.022 Please cite this article as: Erttmann, S. F.; et Biol. Med. (2011), doi:10.1016/j.freeradbiom e activity [4,5]. Thus, the ction of cell-membrane nd involvement in the revealed that the LPS- and IFN-γ-mediated Prx 6 gene induction is in addition to a NO-dependent mechanism, regulated in a NO-indepen- dent manner by both COX-1 and COX-2 [10]. functions proposed for Prx 6 include prote phospholipids against oxidative damage a Peroxiredoxin PI3K PKC Prostaglandin Free radicals Peroxiredoxins (Prxs) are a wid peroxidases that use thiols to reduc range of organic hydroperoxides, and have been classified as typical two-cy two-cysteine Prx (Prx 5), and one-cys number of conserved cysteines and con catalysis [1–3]. Among the sixmammal is the only enzyme that possesses acid lipase A activity in addition to its per © 2011 Elsevier Inc. All rights reserved. tributed superfamily of ogen peroxide, a broad ynitrite. These enzymes Prxs (Prx 1–4), atypical rx (Prx 6) based on the tly by the mechanism of bers of this family, Prx 6 -independent phospho- enriched in the lung compared to other organs and is expressed at highest levels in alveolar epithelial type II cells, bronchiolar Clara cells, and alveolar macrophages [6–8]. A recent study by Diet et al. [9] and own previous work [10] indicated that expression of Prx 6 is increased byLPS and IFN-γ inmurinebonemarrow-derivedmacrophages (BMM). Moreover, we could demonstrate that the LPS- and IFN-γ-induced cyclooxygenase-2 (COX-2) expression and secretion of prostaglandin E2 (PGE2) leads to increased Prx 6 gene expression. Inhibition experiments enzymes, such a cyclopentenone of PGD2, has bee including anti-in estingly, a prev demonstrated th Prx 1 [13]. honate; MAPK, mitogen-activat- ated factor 2; PG, prostaglandin; ted receptor; Prx, peroxiredoxin; thiocyanato-4-(methylsulfinyl) t). l rights reserved. al., PGD2 and PGE2 regulate gene expression o ed.2011.05.022 criptional induction. Nrf2 PPARγ, we found that Nrf2, but not PPARγ, is involved in the PG-dependent increase in Prx 6 mRNA expression. In summary, our data suggest multiple signaling pathways of Prx 6 regulation by PGs and Macrophage MAPK dependent Prx 6 induction. a b s t r a c ta r t i c l e i n f o Article history: Received 25 November 2010 Revised 16 May 2011 Accepted 19 May 2011 Available online xxxx Keywords: Adenylate cyclase JAK2 Peroxiredoxin 6 (Prx 6) i independent phospholipase derived macrophages to LP regulating Prx 6 mRNA leve ability to induce gene expr mechanisms. We provide e expression. Treatment with indicated that Prx 6 gene Furthermore, our study rev f Prx 6 in primary macrophages via Nrf2 trin Breitbach a, Reinhard Walther b, Ivo Steinmetz a ld, 17475 Greifswald, Germany f Greifswald, 17475 Greifswald, Germany bifunctional enzyme with both glutathione peroxidase and acidic Ca2+- activities. We have recently shown that exposure of murine bone marrow- d IFN-γ leads to induction of COX-2 expression and secretion of PGE2, up- his study was designed to investigate various prostaglandins (PGs) for their on of Prxs, in particular Prx 6, and to determine the underlying regulatory ence that both conventional and cyclopentenone PGs enhance Prx 6 mRNA ther activators or inhibitors of adenylate cyclase as well as cAMP analogs pression is regulated by adenylate cyclase in response to PGD2 or PGE2. ed that JAK2, PI3K, PKC, and p38 MAPK contribute to the PGD2- or PGE2- gy & Medicine r.com/ locate / f reeradb iomed s PGD synthase and PGE synthase, respectively. The prostaglandin 15d-PGJ2, the dehydration end product n known to display multifaceted cellular functions, flammatory and cytoprotective effects [11,12]. Inter- ious study using mouse peritoneal macrophages at 15d-PGJ2 is able to increase gene expression of f Prx 6 in primary macrophages via Nrf2, Free Radic. caffeic acid phenethyl ester (CAPE), tert-butylhydroquinone (tBHQ), triglitazone, ciglitazone, GW-9662, and L-NIL were obtained from (Department of Medicine, University of California at San Francisco, San Francisco, USA). 2 S.F. Erttmann et al. / Free Radical Biology & Medicine xxx (2011) xxx–xxx BMMs were generated and cultivated in a serum-free cell culture system as recently described [21]. Briefly, tibias and femurs were aseptically removed and bone marrow cells were flushed with sterile phosphate-buffered saline (PBS) and centrifuged at 150 g for 15 min. The cells were resuspended in RPMI medium containing 5% Panexin BMM (PAN Biotech, Aidenbach, Germany), 2 ng/ml recombinant murine GM-CSF (PAN Biotech), and 50 μM mercaptoethanol and cultivated for at least 10 days at 37 °C in a humidified atmosphere containing 95% air and 5% CO2. Twenty-four hours before stimulation experiments, 2.5×105 BMMs were seeded in 12-well plates. When indicated, BMMs were preincubated for 1 h with appropriate in- hibitors followed by treatment with prostaglandins, LPS, and IFN-γ or corresponding vehicle. The final concentrations of the vehicles were 0.1% for dimethyl sulfoxide (DMSO), ≤0.32% for methyl acetate, and Enzo Life Sciences (Lörrach, Germany). PGD2 and PGE1 were from Calbiochem (Darmstadt, Germany). Griess reagent (modified) and LPS (Escherichia coli serotype 055:B5) were from Sigma–Aldrich (Taufkirchen, Germany) and mIFN-γ was from Roche (Mannheim, Germany). 6-Bnz-cAMP and 8-pCPT-2′-O-Me-cAMP were purchased from Biolog Life Science Institute (Bremen, Germany). Polyclonal antibody against GAPDH and monoclonal antibody against Prx 6 were from AbFrontier (Acris Antibodies, Herford, Germany), polyclonal antibody against Nrf2 (IF) was from Santa Cruz Biotechnology (Heidelberg, Germany) and against Nrf2 (WB) was from Bioworld Technology (St. Louis Park, MN, USA). Cy2-conjugated anti-rabbit IgG was from Dianova (Hamburg, Germany), polyclonal antibodies against histone H3 and horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG were purchased from Cell Signaling (Frankfurt am Main, Germany). Generation, cultivation, and stimulation of BMM C57BL/6 mice were obtained from Charles River (Wiga Sulzfeld, Germany) and C57B/SV129 Nrf2 knockout mice as well as C57B/SV129 wild-typemice [20] were kindly provided by Thomas Herdegen (Institute of Experimental and Clinical Pharmacology, University of Kiel, Germany), with permission of Yuet W. Kan PGD2 and PGE2 exert their effects through different G-protein- coupled receptors, DP1-2 or EP1-4 [14]. Among these, the DP1, EP2, and EP4 receptors increase cAMP via activation of adenylate cyclase [15]. To date, most cAMP-mediated effects of PGD2 or PGE2 have been explained by the classic downstream target, protein kinase A (PKA), which phosphorylates the cAMP response element-binding protein (CREB) in a variety of mammalian cells [16,17], or by a novel target for cAMP, exchange protein directly activated by cAMP (Epac) [18,19]. So far, the regulatory mechanisms controlling Prx 6 expression in response to prostaglandins are not known. Thus, the aim of this study was first to investigate gene expression of peroxiredoxins in primary macrophages by conventional and cyclopentenone prostaglandins and second to characterize the role of various protein kinases and transcription factors on Prx 6 mRNA expression. Materials and methods Materials PGA1, PGA2, PGE2, PGF2α, 15d-PGJ2, dibuturyl-cAMP, forskolin, IBMX, H-89, KT5720, MDL 12,330A, LY294002, Ro31-8220, Gö6796, SB202190, SP600125, PD98059, AG490, MAFP, AACOCF3, sulforaphane, ≤0.17% for acetone. Please cite this article as: Erttmann, S. F.; et al., PGD2 and PGE2 regulate g Biol. Med. (2011), doi:10.1016/j.freeradbiomed.2011.05.022 Nitrite assay Nitrite, the stable end product of nitric oxide, was quantified in culture medium using the Griess reagent (modified). Briefly, 500 μl of each supernatant was mixed with the same volume of Griess reagent and absorbance was measured at 540 nm after 15 min. Nitrite concentration was determined from a sodium nitrite standard curve. RNA isolation and quantitative real-time PCR (qRT-PCR) Total RNA was isolated using the TRIzol reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer's instructions. Reverse transcription of 1 μg of total RNA was performed using the Moloney murine leukemia virus reverse transcriptase (Promega, Mannheim, Germany) and 0.5 μg oligo(dT) primer (Invitrogen). qRT- PCR was performed using the LightCycler 480, and detection of amplification products was done using the LightCycler 480 Probes Master Kit (Roche). TaqMan PCR probes and gene-specific primer pairs were generated by Microsynth (Balgach, Switzerland). Data were analyzed with LightCycler software version 1.5. The reference gene RPLP0 served for the standardization of the individual PCRs. All assays were performed in duplicate and repeated four to six times as indicated. Western blot analysis Proteins were prepared using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Protein content was determined using the Bradford method. Equal amounts of protein were separated by SDS–PAGE and transferred onto nitrocellulose membranes by electroblotting. Membranes were blocked with 1× Roti-Block (Roth, Karlsruhe, Germany) for 1 h at room temperature and subsequently incubated overnight at 4 °C with a rabbit anti- GAPDH, mouse anti-Prx 6, rabbit anti-Nrf2, or rabbit anti-histone H3 antibody (in 20 mM Tris, 138 mM NaCl, pH 7.6, 5% (w/v) bovine serum albumin (BSA), 0.1% (v/v) Tween 20). HRP-conjugated anti- mouse or anti-rabbit IgG (in 1× Roti-Block) was used as a secondary antibody for 1 h at room temperature. The LumiGLO system (Cell Signaling) was used for detection. All experiments were performed at least twice. Densitometric measurements for relative quantification were done using Kodak software. Immunofluorescence staining Nuclei of living primarymacrophages, cultured on coverslips, were stained with the blue fluorescent Hoechst 33342 dye (Invitrogen) for 10 min at 37 °C. Macrophages were washed with ice-cold PBS, incubated for 10 min in ice-cold methanol, and washed three times with IF buffer (0.2% (w/v) BSA, 0.05% (w/v) saponin, 0.1% (w/v) sodium azide in PBS, pH 7.4). To block nonspecific antibody binding, cells were incubated for up to 1 h in IF buffer followed by an overnight incubation at 4 °C in a humidity chamber with polyclonal rabbit anti- Nrf2 antibody (in IF buffer). After a wash in IF buffer, the immunoreacted primary antibody was visualized with green fluores- cent Cy2-conjugated goat anti-rabbit IgG (in IF buffer) by incubation for 1 h at room temperature in the dark. After another wash in IF buffer, slices were covered with Fluoprep (bioMérieux, Nürtingen, Germany) and observed by fluorescence microscopy with a BZ-9000 microscope (Keyence Corp., Neu-Isenburg, Germany). Preparation of nuclear extracts Cells were harvested in ice-cold Dulbecco's PBS (D-PBS; Invitrogen). After beingwashed, the cellswere incubated in hypomolarHepes buffer (10 mM Hepes, pH 7.6, 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, Complete Mini EDTA-free protease inhibitor cocktail ene expression of Prx 6 in primary macrophages via Nrf2, Free Radic. (Roche)) for 10 min on ice. Cells were lysed by the addition of NP-40. After thenucleiwere collectedbycentrifugation, the supernatant,which contained the cytosolic proteins, was recovered. The pelleted nuclei were rinsed with ice-cold D-PBS and nuclear proteins were extracted subsequently inhypermolarHepesbuffer (20 mMHepes, pH7.9, 0.42 M NaCl, 25% glycerol (v/v), 0.2 mMEDTA, 0.5 mMdithiothreitol, Complete Mini EDTA-free protease inhibitor cocktail (Roche)) and agitated for 15 min at 4 °C. Statistical analysis Statistical analyses were performed using GraphPad Prism version 5.0. All data are expressed as means of duplicate determinations from individual experiments and are presented as the mean±SEM, where n≥4 experiments. Comparison of groups was performed using one- way ANOVA nonparametric test followed by the Bonferroni posttest for multiple comparisons or using Kruskal–Wallis test followed by the Dunn posttest. Pb0.05 was considered statistically significant. Results Gene expression of Prx 6 is increased by prostaglandins in bone marrow-derived macrophages To elucidate the role of PGs in Prx gene expression, BMMs of C57BL/6 mice were stimulated with conventional PGs, i.e., PGD2 (50 μM), PGE1 (50 μM), PGE2 (50 μM), PGF2α (50 μM); cyclopentenone PGs, i.e., PGA1 (50 μM), PGA2 (50 μM), 15d-PGJ2 (10 μM); or corresponding vehicle (DMSO,methyl acetate). After 18 h of stimulation, nitritewasquantified and mRNA expression of Prxs 1–6 was measured by qRT-PCR. In agreement with Itoh et al. [13], gene expression of Prx 1 was significantly increased by 15d-PGJ2, whereas Prx 2, 3, and 4 mRNA levels were not changed or even decreased by PGs.Moreover, Prx 5was slightly increased by PGE1, PGE2, and PGF2α. In contrast, Prx 6 was enhanced by all PGs used in this study, most notably PGD2, PGE2, PGA1, and 15d-PGJ2 (Fig. 1). Nitrite was not changed by any of the PGs (data not shown), and PG-induced Prx 6 gene expression was not altered by the iNOS inhibitor L-NIL (Supplementary Fig. 1), indicating that induction of Prxs by PGs is NO-independent. These results imply that in addition to the selective induction of Prx 1 gene expression by 15d-PGJ2, mRNA expression of Prx 5 is slightly enhanced by PGs of the E and F classes, whereas Prx 6 transcription is strongly induced by various PGs in primary murine macrophages. Prx 6 mRNA expression is time- and dose-dependently regulated by cyclopentenone prostaglandins PGA1, PGA2, and 15d-PGJ2 We then performed both dose–response and time-course exper- iments on Prx 6 gene expression in response to cyclopentenone prostaglandins. Therefore, BMMs were cultured for 18 h in the presence of increasing concentrations of PGA1 (12.5–50 μM), PGA2 (12.5–50 μM), or 15d-PGJ2 (2.5–10 μM). As shown in Fig. 2, Prx 6 expression was induced at the mRNA level by concentrations of PGA1 and PGA2 in the range of 12.5–50 μMand 15d-PGJ2 in the range of 2.5– - - we hicl P0, i 3S.F. Erttmann et al. / Free Radical Biology & Medicine xxx (2011) xxx–xxx A1 A2 J2E2D2 F2αE1 - A1 A2 J2E2D2 F2αE1 - A1 A2 J2E2D2 F2αE1 Fig. 1. Prostaglandins increase mRNA expression of Prxs in primary macrophages. BMM PGE1 (50 μM), PGE2 (50 μM), PGF2α (50 μM), or 15d-PGJ2 (10 μM) or corresponding ve quantitative real-time PCR. The fold difference in mRNA expression, normalized to RPL performed using one-way ANOVA followed by the Bonferroni posttest (*Pb0.05, **Pb0.01, Please cite this article as: Erttmann, S. F.; et al., PGD2 and PGE2 regulate g Biol. Med. (2011), doi:10.1016/j.freeradbiomed.2011.05.022 A1 A2 J2E2D2 F2αE1 - A1 A2 J2E2D2 F2αE1 - A1 A2 J2E2D2 F2αE1 PG PG PG re cultured in the presence or absence of PGA1 (50 μM), PGA2 (50 μM), PGD2 (50 μM), e (DMSO, methyl acetate) for 18 h. RNA was analyzed for Prx 1–6 gene expression by s indicated. Data are presented as means with SEM (n=6). Comparison of groups was ***Pb0.001). ene expression of Prx 6 in primary macrophages via Nrf2, Free Radic. 4 S.F. Erttmann et al. / Free Radical Biology & Medicine xxx (2011) xxx–xxx B - 12.5 25 50 PGA1 A 10 μM (Figs. 2A–C). Furthermore, time-course experiments (9–24 h) revealed that gene expression of Prx 6 is maximally induced after 18 h in response to PGA1 and 15d-PGJ2 and after 24 h in response to PGA2 (Figs. 2D–F). PGD2 and PGE2 increase Prx 6 mRNA expression in a dose- and time-dependent manner In addition, we performed a dose- and time-dependent analysis of Prx 6 mRNA expression after stimulation with conventional prosta- glandins. BMMs were cultured for 18 h in the presence of increasing concentrations (12.5–50 μM) of PGD2 or PGE2. As shown in Figs. 3A and D, treatment with both PGs resulted in significantly higher Prx 6 mRNA levels, depending on the concentration used. A sixfold (PGD2) or fourfold (PGE2) increase was reached with 50 μM corresponding PG. Subsequent time-course experiments (9–24 h) revealed that PGD2 elicited Prx 6 gene induction 9 h after stimulation, with a - 2.5 5 10 15d-PGJ2 C - 12.5 25 50 PGA2 Fig. 2. Gene expression of Prx 6 is time- and dose-dependently regulated by PGA1, PGA2, o (C) 15d-PGJ2 at the indicated concentrations (12.5–50 μM; 2.5–10 μM) for 18 h. BMMs were PGJ2 (10 μM) for 9–24 h. RNA was analyzed for Prx 6 gene expression by quantitative real-tim are presented as means with SEM (n≥4). Statistical analysis was performed using Kruskal– Please cite this article as: Erttmann, S. F.; et al., PGD2 and PGE2 regulate g Biol. Med. (2011), doi:10.1016/j.freeradbiomed.2011.05.022 E - 9 12 18 PGA1 24 D [h] maximum after 18 h (Fig. 3B). In contrast, mRNA levels of Prx 6 were increased by PGE2 from 18 to 24 h (Fig. 3E). The following gene expression experiments were conducted at 18 h after stimulation with 50 μM PGD2 or PGE2. To determine whether the Prx 6 mRNA induction in PGD2- and PGE2-stimulated BMMs results in an enhanced protein amount, we performed Western blot analyses. Protein expression of Prx 6 was time-dependently increased in response to PGD2 with a maximum after 12–18 h (Fig. 3C) and PGE2 with a maximum after 48 h (Fig. 3F). Adenylate cyclase is involved in the up-regulation of the Prx 6 gene in response to PGD2 and PGE2 To study the molecular signaling mechanisms implicated in the PGD2- and PGE2-dependent Prx 6mRNA induction, we first elucidated the role of adenyl