The Anti-inflammatory Effects of Selenium Are Mediated through 15-Deoxy-Δ12,14-prostaglandin J2 in Macrophages
2007; Elsevier BV; Volume: 282; Issue: 25 Linguagem: Inglês
10.1074/jbc.m703075200
ISSN1083-351X
AutoresHema Vunta, Faith B. Davis, Umamaheswari D. Palempalli, Deepa Bhat, Ryan J. Arner, Jerry T. Thompson, Devin G. Peterson, C. Channa Reddy, K. Sandeep Prabhu,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoSelenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-κB-dependent pro-inflammatory gene expression. To understand the molecular mechanisms underlying the anti-inflammatory property of selenium, we examined the activity of a key kinase of the NF-κB cascade, IκB-kinase β (IKKβ) subunit, as a function of cellular selenium status in murine primary bone marrow-derived macrophages and RAW264.7 macrophage-like cell line. In vitro kinase assays revealed that selenium supplementation decreased the activity of IKKβ in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of selenium-supplemented macrophages resulted in a time-dependent increase in 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) formation, an endogenous inhibitor of IKKβ activity. Further analysis revealed that inhibition of IKKβ activity in selenium-supplemented cells correlated with the Michael addition product of 15d-PGJ2 with Cys-179 of IKKβ, while the formation of such an adduct was significantly decreased in the selenium-deficient macrophages. In addition, anti-inflammatory activities of selenium were also mediated by the 15d-PGJ2-dependent activation of the peroxisome proliferator-activated nuclear receptor-γ in macrophages. Experiments using specific cyclooxygenase (COX) inhibitors and genetic knockdown approaches indicated that COX-1, and not the COX-2 pathway, was responsible for the increased synthesis of 15d-PGJ2 in selenium-supplemented macrophages. Taken together, our results suggest that selenium supplementation increases the production of 15d-PGJ2 as an adaptive response to protect cells against oxidative stress-induced pro-inflammatory gene expression. More specifically, modification of protein thiols by 15d-PGJ2 represents a previously undescribed code for redox regulation of gene expression by selenium. Selenium is an essential micronutrient that suppresses the redox-sensitive transcription factor NF-κB-dependent pro-inflammatory gene expression. To understand the molecular mechanisms underlying the anti-inflammatory property of selenium, we examined the activity of a key kinase of the NF-κB cascade, IκB-kinase β (IKKβ) subunit, as a function of cellular selenium status in murine primary bone marrow-derived macrophages and RAW264.7 macrophage-like cell line. In vitro kinase assays revealed that selenium supplementation decreased the activity of IKKβ in lipopolysaccharide (LPS)-treated macrophages. Stimulation by LPS of selenium-supplemented macrophages resulted in a time-dependent increase in 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) formation, an endogenous inhibitor of IKKβ activity. Further analysis revealed that inhibition of IKKβ activity in selenium-supplemented cells correlated with the Michael addition product of 15d-PGJ2 with Cys-179 of IKKβ, while the formation of such an adduct was significantly decreased in the selenium-deficient macrophages. In addition, anti-inflammatory activities of selenium were also mediated by the 15d-PGJ2-dependent activation of the peroxisome proliferator-activated nuclear receptor-γ in macrophages. Experiments using specific cyclooxygenase (COX) inhibitors and genetic knockdown approaches indicated that COX-1, and not the COX-2 pathway, was responsible for the increased synthesis of 15d-PGJ2 in selenium-supplemented macrophages. Taken together, our results suggest that selenium supplementation increases the production of 15d-PGJ2 as an adaptive response to protect cells against oxidative stress-induced pro-inflammatory gene expression. More specifically, modification of protein thiols by 15d-PGJ2 represents a previously undescribed code for redox regulation of gene expression by selenium. Macrophages play central roles as effector cells in inflammatory reactions and cell-mediated immune responses. While performing these functions, these cells produce such reactive oxygen species in the form of superoxide anion, hydrogen peroxide, hydroxyl, and lipid peroxyl radicals along with a great number of pro-inflammatory substances, including complement components, PGs, 3The abbreviations used are: PG, prostaglandin; GPX, glutathione peroxidase; HIV, human immunodeficiency virus; NF-κB, nuclear factor-κB; COX, cyclooxygenase; IKKβ, IκB-kinase β; 15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; PPAR, peroxisome proliferator-activated receptor; H-PGDS, hematopoietic PGD2 synthase; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; BMDM, bone marrow-derived macrophage; GST, glutathione S-transferase; GW9662, 2-chloro-5-nitro-N-phenylbenzamide; HQL-79, 4-(diphenylmethoxy)-1-[3-(1H-tetrazol-5-yl)propylpiperidine]; LC-MS, liquid chromatography-mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. chemokines, and cytokines like interleukin-1β and tumor necrosis factor-α (1Speer C.P. Gahr M. Monatsschr. Kinderheilkd. 1989; 137: 390-395PubMed Google Scholar). Such reactions represent a potentially toxic insult, which if not counteracted, will lead to membrane dysfunction, DNA damage and inactivation of proteins, leading to the onset and/or progression of many disease pathologies (2Halliwell B. Guteridge J.M.C. Free Radicals in Biology and Medicine. Clarendon Press, Oxford1989Google Scholar, 3Spector A. J. Ocul. Pharmacol. Ther. 2000; 16: 193-201Crossref PubMed Scopus (210) Google Scholar, 4Toyokuni S. Pathol. Int. 1999; 49: 91-102Crossref PubMed Scopus (490) Google Scholar). In contrast to the conventional dogma that reactive oxygen species are mostly triggers for oxidative damage of biological structures, it is now increasingly clear that physiologically relevant concentrations of reactive oxygen species can regulate a variety of key molecular pathways that may be linked with important cell functions, including gene expression (5Sen C.K. Packer L. 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In recent years, the role of selenium in preventing human disease has gained new attention following the association of "super supplementation" with decreased incidences of prostate cancer in a few preliminary studies (11Gasparian A.V. Yao Y.J. Lu J. Yemelyanov A.Y. Lyakh L.A. Slaga T.J. Budunova I.V. Mol. Cancer Ther. 2002; 1: 1079-1087PubMed Google Scholar, 12Vogt T.M. Ziegler R.G. Graubard B.I. Swanson C.A. Greenberg R.S. Schoenberg J.B. Swanson G.M. Hayes R.B. Mayne S.T. Int. J. Cancer. 2003; 20: 664-670Crossref Scopus (104) Google Scholar, 13Sonn G.A. Aronson W. Litwin M.S. Prostate Cancer Prostatic Dis. 2005; 8: 304-310Crossref PubMed Scopus (142) Google Scholar). It has been proposed that selenium prevents malignant transformation of cells by serving as a "redox switch" through its role in catalyzing oxidation-reduction reactions of critical thiol groups or disulfide bonds, possibly through selenoproteins (14Hadley K.B. Sunde R.A. J. Nutr. 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Although both IKKα and IKKβ can phosphorylate all three IκB proteins in vitro, studies in mice that are deficient in IKK subunits show that, in most cells, IKKβ has the dominant role in signal-induced phosphorylation and degradation of these proteins (21Karin M. Yamamoto Y. Wang Q.M. Nat. Rev. Drug Discov. 2004; 3: 17-26Crossref PubMed Scopus (1243) Google Scholar, 22Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2079) Google Scholar). In addition to phosphorylation by upstream kinases, the enzymatic activity of IKKβ is subjected to further control by Michael adduct formation with α,β-unsaturated carbonyl compounds, 4-hydroxynonenal (23Ji C. Kozak K.R. Marnett L.J. J. Biol. Chem. 2001; 276: 18223-18228Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) or cyclopentenone PGs such as PGA1 and 15d-PGJ2, with a critical cysteine (Cys-179) residue in the activation loop (24Rossi A. Kapahi P. Natoli G. Takahashi T. 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Therefore, cellular production of 15d-PGJ2 may mediate anti-inflammatory responses via these pathways. However, there are no reports on the preferential increase in the intracellular levels of 15d-PGJ2 to support these claims. Here, we show for the first time that selenium supplementation of macrophages leads to the preferential increase in 15d-PGJ2 via the arachidonic acid oxidation by COX-1, rather than COX-2. In macrophages and other immune cells, PGH2 is further converted to PGD2 by the hematopoietic PGD2 synthase (H-PGDS) that undergoes two spontaneous non-enzymatic dehydration reactions to form 15d-PGJ2 (29Shibata T. Kondo M. Osawa T. Shibata N. Kobayashi M. Uchida K. J. Biol. Chem. 2002; 277: 10459-10466Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). The present study is based on the hypothesis that the anti-inflammatory role of selenium occurs, in part, via 15d-PGJ2-dependent intracellular signaling pathways in macrophages and that COX-1 plays a pivotal role in the control of NF-κB activity. Reagents—Bacterial endotoxin lipopolysaccharide (LPS), sodium selenite, and GW9662 were from Sigma. Anti-COX-1, anti-COX-2, COX inhibitors, indomethacin, SC-560 (for COX-1), and CAY10404 (for COX-2) were obtained from Cayman Chemicals (Ann Arbor, MI). Antibodies for IKKα, pIκBα, glyceraldehyde-3-phosphate dehydrogenase, and p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-IKKβ was from Imgenex (San Diego, CA). Anti-15d-PGJ2 was from Assay Designs (Ann Arbor, MI). Goat anti-rabbit IgG, anti-mouse IgG conjugated to horseradish peroxidase, polyvinylidene difluoride, and West Pico chemiluminescence reagents were purchased from Pierce. Mouse IKKβ-(173–186) was synthesized at the Macromolecular core facility, Pennsylvania State College of Medicine, Hershey, PA. Cell Culture and Stimulation—The murine macrophage cell line RAW264.7 (ATCC) was cultured in Dulbecco's modified Eagle's medium containing 5% defined fetal bovine serum (HyClone), 80 μg/ml gentamicin, and 2 mm l-glutamine (Invitrogen) at 37 °C with a 5% CO2/air mixture. Total selenium in the fetal bovine serum was quantitated to be 6 pmol/ml. Cells were cultured in Dulbecco's modified Eagle's medium either supplemented with selenium (2 nmol/ml of media) or without any added as described from our laboratory (17Zamamiri-Davis F. Lu Y. Thompson J.T. Prabhu K.S. Reddy P.V. Sordillo L.M. Reddy C.C. Free Radic. Biol. Med. 2002; 32: 890-897Crossref PubMed Scopus (85) Google Scholar). Cell viability and growth rates of selenium-supplemented cells were similar to their selenium-deficient counterparts. The enzymatic activity was used as a marker of cellular selenium status (9Allan C.B. Lacourciere G.M. Stadtman T.C. Annu. Rev. Nutr. 1999; 19: 1-16Crossref PubMed Scopus (295) Google Scholar). About 1 × 106 selenium-deficient and selenium-supplemented cells were seeded in a 6-well plate, and then cultured in respective media for ∼24 h to allow the cell number to approximately double. Cells were stimulated with LPS (0–1 μg/ml) and/or other compounds for the indicated time periods. Upon treatment, the cells were harvested, washed with cold sterile PBS and stored at –80 °C until further use. Femoral bone marrow plugs, from mice maintained on a selenium-deficient or a selenium-supplemented diet, were isolated, and adherent cells, hereafter referred to as primary bone marrow-derived macrophages (BMDMs), were differentiated in their respective media containing 20% L929 fibroblast media supernatant (as a source of granulocyte-macrophage colony-stimulating factor) for 1 week. The L929 cells were also cultured under selenium-deficient or selenium-supplemented conditions. The selenium-deficient and selenium-supplemented diets were formulated based on an American Institute of Nutrition recommended rodent diet containing 0.01 or 0.4 ppm of selenium, as described (30Moskovitz J. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7486-7490Crossref PubMed Scopus (92) Google Scholar). The diets were purchased from Zeigler (Gardners, PA). The BMDM cultures from selenium-deficient and selenium-supplemented mice were used in all experiments. LPS Treatment of Mice—Selenium-deficient and selenium-supplemented mice (n = 3 in each category) were treated with Escherichia coli serotype 0111:B4 LPS (at 5 mg/kg body weight) or PBS control by intraperitoneal injection. The mice were euthanized after 6 h of injection. The serum from each mouse was prepared and used in 15d-PGJ2 enzyme-linked immunosorbent assays as described later in this section. All animal protocols were approved by the Institutional Animal Care and Use Committee. Preparation of Cell Lysates—The frozen cell pellet was resuspended in 50 μl of mammalian protein extraction reagent (M-PER, Pierce) containing 1 mm EDTA, 10 μm leupeptin, and 1 mm phenylmethylsulfonic acid for 30 min on ice with intermittent vortexing. Supernatants were prepared by centrifuging the cell lysate at 10,000 × g for 15 min at 4 °C and used for analyses. Protein concentration in the cell supernatants was determined by BCA protein assay (Pierce). Electrophoresis and Immunoblotting—Thirty micrograms of protein from RAW264.7 or BMDM cell lysates was separated on a 12.5% SDS-polyacrylamide gel and transblotted onto polyvinylidene difluoride membrane as described (31Prabhu K.S. Arner R.J. Vunta H. Reddy C.C. J. Biol. Chem. 2005; 280: 19895-19901Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The membrane was blocked with Tris-buffered saline containing 0.05% Tween 20 and 5% skim milk (w/v). The membrane was probed with primary antibody followed by an appropriate secondary antibody coupled to horseradish peroxidase. Immunoprecipitation—Cell lysates (∼30–50 μg of protein) from LPS-treated and untreated selenium-deficient or selenium-supplemented RAW264.7 and BMDMs were used with IKKα- (Santa Cruz Biotechnology) or IKKβ-specific IgG (Imgenex). The immunoprecipitated IKK·IgG complex was pulled down using Protein A/G-Sepharose (GE Amersham Biosciences) and used in Western blot analysis, as described above, or in vitro kinase assays, as described below. The membrane was probed with anti-15d-PGJ2. [14C]Arachidonic Acid Treatment of Macrophages—The selenium-deficient and selenium-supplemented RAW264.7 cells were pretreated with [1-14C]arachidonic acid (1 μCi and 30 μm, American Radiochemicals, St. Louis, MO) for 2 h prior to stimulation with LPS for 1 h. The cell lysates were used to isolate IKKβ by immunoprecipitation, as described earlier, and analyzed on an SDS-PAGE followed by autoradiography. The blots were reprobed for IKKβ to confirm near-equal immunoprecipitation. In Vitro Kinase Assays—Cell lysates for in vitro kinase assays were prepared using the lysis buffer as described (32Carcamo J.M. Pedraza A. Borquez-Ojeda O. Golde D.W. Biochemistry. 2002; 41: 12995-13002Crossref PubMed Scopus (218) Google Scholar). In the case of IKK, the cell lysate (∼30–50 μg of protein) was incubated with GST-IκBα and ATP for 1 h in the kinase wash buffer (50 mm Tris-Cl, pH 8.0, containing 100 μm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 10 mm β-glycerophosphate, 10 mm NaF, and 1 mm sodium vanadate) at 25 °C. Glutathione-Sepharose beads (Amersham Biosciences) were added to the reaction mixture, and the mixture was centrifuged. The pellet was washed three times with sterile PBS. The phospho-labeled GST-IκBα was separated by SDS-PAGE and immunoblotted onto polyvinylidene difluoride membranes. The membrane was probed with anti-phosphoserine (Sigma). Furthermore, to quantitate the activity, immunoprecipitated IKKα or IKKβ was incubated with [32P]ATP (2 μCi, Amersham Biosciences) and GST-IκBα fusion protein as described (32Carcamo J.M. Pedraza A. Borquez-Ojeda O. Golde D.W. Biochemistry. 2002; 41: 12995-13002Crossref PubMed Scopus (218) Google Scholar). The reaction mixture was subjected to gel-filtration chromatography using the Bio-Gel P30 pre-packed columns (Bio-Rad). The flow-through, which contained the GST-[32P]IκBα, was subjected to liquid scintillation counting. Chemical Treatments—Selenium-deficient and selenium-supplemented RAW264.7 and BMDM cells (1 × 106) were seeded into each well of a 6-well plate and treated with 1 μm indomethacin or SC-560 to inhibit COX-1. 10 nm CAY10404 was used as a COX-2-specific inhibitor. In all cases, the treatment with inhibitors (for 24 h) was followed by stimulation with LPS for the indicated time periods. The cell lysates were prepared as described earlier and subjected to Western immunoblot analyses, in vitro kinase assays, while the media supernatants were used for 15d-PGJ2 assays. An irreversible PPARγ antagonist, 2-chloro-5-nitro-N-phenylbenzamide (GW9662, 1 μm) was used in some studies to determine the specific role of 15d-PGJ2-dependent activation of PPARγ. The cells were pretreated with GW9662 for 12 h prior to LPS stimulation. To inhibit the activity of H-PGDS, selenium-supplemented cells were pretreated with 50 μm 4-(diphenylmethoxy)-1-[3-(1H-tetrazol-5-yl)propylpiperidine] (HQL-79, Cayman), prior to LPS stimulation for 12 h. Supernatants were used in 15d-PGJ2 analysis as described below. All chemical treatment studies included an appropriate Me2SO4 vehicle control. Quantitation of PGD2 and 15d-PGJ2—A 96-well-based enzyme immunoassay kit, from both Cayman Chemicals and Assay Designs (Ann Arbor, MI), was used to quantitate PGD2 and 15d-PGJ2, respectively. The concentrations of 15d-PGJ2 in culture media supernatants of RAW264.7 and BMDM in the presence or absence of LPS (0–1 μg/ml) for various time periods were determined by enzyme immunoassay according to the manufacturer's instructions and normalized to total cellular protein. PGD2 was derivatized using methoximylamine-HCl as per the recommendation of the supplier. Standard calibration curves were prepared using PGD2 methoxime or 15d-PGJ2 and fitted to a log-linear, logit, multiway frequency regression analysis. PGD2 and 15d-PGJ2 were quantitated in cell lysates and normalized to total protein in the cell lysates. LC-MS Analysis of 15d-PGJ2 Production—Culture media supernatants from LPS-stimulated selenium-deficient and selenium-supplemented macrophages were acidified with 2 n HCl and clarified by centrifugation at 10,000 × g for 5 min. Supernatants were processed using a C18-Sep-Pak column cartridge (Waters), and bound 15d-PGJ2 was eluted with methanol, evaporated, and stored in ethyl acetate at –80 °C until further analysis. As an internal control to calculate extraction efficiency, 200 ng of deuterated 15d-PGJ2(d4) (Cayman Chemicals) was added to the supernatants before extraction. The 15d-PGJ2 was resolved on a Restek Ultra Aqueous C18 (5 μm, 250 mm × 2.1 mm) high-performance liquid chromatography column (Bellefonte, PA) on an aqueous acetonitrile gradient with 0.1% formic acid at a flow rate of 0.2 ml/min. MS analysis was performed on a Micromass ZMD mass spectrometer (Waters) set to scan mode (m/z 200–400) for authentic standards, whereas selective ion monitoring set to m/z 317 (M-H+) and 321 (M-H+) for 15d-PGJ2(d0) and 15d-PGJ2(d4), respectively, was used for quantitation in the samples. Standard calibration curves for 15d-PGJ2(d0) and 15d-PGJ2(d4) were set up for the quantitation and calculations were performed based on the following: b(0) = 6545.09, b(1) = 25.51, and r2 = 0.99 (for d0) and b(0) = 253.48, b(1) = 79.646, and r2 = 0.98 (for d4). Mass Spectrometric Analysis of Post-translational Modification of IKKβ—To further confirm the modification of IKKβ by 15d-PGJ2 and support the immunoprecipitation studies, murine IKKβ peptide-(173–186), LDQGSLCTSFVGTL, was incubated with Me2SO, authentic 15d-PGJ2 (mol/mol), or total lipid extract from selenium-supplemented macrophages (LPS-treated for 2 h) for 30 min at 37 °C in PBS. The samples were analyzed by MALDI-TOF-MS for modification. Transient Transfection Assays—Murine COX-2 gene promoter (–2000 to +75) and its NF-κB double mutant in pGL3 luciferase vector were prepared as described (17Zamamiri-Davis F. Lu Y. Thompson J.T. Prabhu K.S. Reddy P.V. Sordillo L.M. Reddy C.C. Free Radic. Biol. Med. 2002; 32: 890-897Crossref PubMed Scopus (85) Google Scholar). The plasmid constructs were transfected into RAW264.7 cells using FuGENE 6 transfection reagent as per the instructions of the supplier (Roche Applied Science). To normalize the transfection efficiency, β-galactosidase activity from the pSV-βGal (Promega, Madison, WI) plasmid and total protein were used. RAW264.7 cells were stimulated with LPS for 4 h post-transfection. Cell lysates were prepared as described above, and the luciferase activity was read in a Turner plate luminometer. siRNA Experiments—The siRNA target sequence for murine COX-1 mRNA was designed using the Dharmacon siGENOME™ design tool available online. The 21-base siRNA oligonucleotides were obtained from Dharmacon and annealed according to the manufacturer's specifications. For transfection, RAW264.7 or BMDM was seeded into 6-well plates (1 × 106 cells/well) without gentamicin. After 24 h, cells were transfected with siRNA duplex using TransIT®-siQUEST™ (Mirus Bio Corp.) according to the manufacturer's specifications. siRNA duplexes were used at a final concentration of 200 pmol/well. To ensure maximum effect, a second transfection was performed after 4 h, and the cells were allowed to recover for an additional 8 h before treatment with LPS. Cells and media supernatants from LPS-treated (12 h) or untreated cells were collected for 15d-PGJ2 quantitation, whereas the corresponding cell lysates were used for IKKβ activity and Western immunoblot analyses. Preparation of Nuclear Extracts for Electrophoretic Mobility Shift Assay—For electrophoretic mobility shift assay experiments, nuclear proteins were isolated as described previously from our laboratory (31Prabhu K.S. Arner R.J. Vunta H. Reddy C.C. J. Biol. Chem. 2005; 280: 19895-19901Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The DNA sequences of the sense strand of double-stranded oligonucleotides specific for NF-κB and PPARγ were 5′-GATCCAGTTGAGGGGACTTTCCCAGGC-3′ and 5′-GGTGAGGAGGGGAAGGGTCAGTGTG-3′, respectively. Complementary strands were annealed, and double-stranded oligonucleotides were labeled with [γ-32P]ATP (3000 Ci/mmol and 10 mCi/ml) using the T4 polynucleotide kinase (New England Biolabs). Five micrograms of nuclear proteins was incubated for 10 min at 4 °C in a binding buffer specific for NF-κB (20 mm Tris-HCl, pH 7.9, 5 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm EDTA, and 20% glycerol) or PPARγ (20 mm HEPES, pH 7.5, 50 mm KCl, 0.175 mm EDTA, and 5% (v/v) glycerol) in the presence of 2 μg of poly(dI-dC). The extracts were then incubated for 30 min at 4 °C with 10,000 cpm of 32P-labeled NF-κB or PPARγ probes. The samples were analyzed as described earlier from our laboratory. NF-κBor PPARγ bands were confirmed by competition with a 100-fold excess of the respective unlabeled probe. Differential Selenium Status in Macrophages—Culturing of RAW264.7 cells in the presence or absence of selenium, as well as primary BMDMs isolated and differentiated from mice maintained on selenium-deficient and selenium-supplemented diets, yielded cell populations that exhibited differential selenium status as seen by cytosolic GPX activity levels (Fig. 1, A and B). A 6-fold difference in the cytosolic GPX activity in the selenium-deficient and selenium-supplemented BMDM was seen. Along the same lines, liver homogenates from selenium-deficient and selenium-supplemented mice demonstrated a 7-fold difference in the enzymatic activity of cytosolic GPX (data not shown). In the case of RAW264.7 macrophages, a difference of ∼12-fold was seen between the two groups (Fig. 1B). The results of differences in GPX activity are consistent with those previously reported in RAW264.7 macrophages from our laboratory (17Zamamiri-Davis F. Lu Y. Thompson J.T. Prabhu K.S. Reddy P.V. Sordillo L.M. Reddy C.C. Free Radic. Biol. Med. 2002; 32: 890-897Crossref PubMed Scopus (85) Google Scholar). Furthermore, the expression of GPX1 were also found to be significantly different in the two groups in that the selenium-supplemented cells demonstrated a higher level of GPX1 expression compared with the selenium-deficient cells (data not shown). Accordingly, the selenium-supplemented and selenium-deficient BMDM and RAW264.7 macrophages were used in all the experiments described below. Selenium Deficiency Exacerbates COX-2 Expression via Increased Levels of pIκBα Leading to the Activation of NF-κB—Stimulation of selenium-deficient RAW264.7 and BMDM cells with LPS for 0–24 h clearly demonstrated exacerbated expression of COX-2 when compared with those cultured in the presence of selenium (Fig. 2, A and B). The differences in expression were obvious as early as 30 min of stimulation of cells (Fig. 2B). Previous reports from our laboratory have indicated that NF-κB is a key transcription factor in the regulation of COX-2 expression in selenium-deficient cells (17Zamamiri-Davis F. Lu Y. Thompson J.T. Prabhu K.S. Reddy P.V. Sordillo L.M. Reddy C.C. Free Radic. Biol. Med. 2002; 32: 890-897Crossref PubMed Scopus (85) Google Scholar). To further understand the role of selenium on the NF-κB-dependent transcription of COX-2, we mutated the two NF-κB sites in the promoter of murine COX-2. As seen in Fig. 2C, the transient transfection studies with wild-type mouse COX-2 promoter luciferase reporter exhibited a 4- to 5-fold increase in activity in selenium-deficient RAW264.7 cells, whereas there was no increase noted in the NF-κB double mutant reporter. The abrogation of luciferas
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