Selenoprotein-dependent Up-regulation of Hematopoietic Prostaglandin D2 Synthase in Macrophages Is Mediated through the Activation of Peroxisome Proliferator-activated Receptor (PPAR) γ*

The plasticity of macrophages is evident from their dual role in inflammation and resolution of inflammation that are accompanied by changes in the transcriptome and metabolome. Along these lines, we have previously demonstrated that the micronutrient selenium increases macrophage production of arachidonic acid (AA)-derived anti-inflammatory 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and decreases the proinflammatory PGE2. Here, we hypothesized that selenium modulated the metabolism of AA by a differential regulation of various prostaglandin (PG) synthases favoring the production of PGD2 metabolites, Δ12-PGJ2 and 15d-PGJ2. A dose-dependent increase in the expression of hematopoietic-PGD2 synthase (H-PGDS) by selenium and a corresponding increase in Δ12-PGJ2 and 15d-PGJ2 in RAW264.7 macrophages and primary bone marrow-derived macrophages was observed. Studies with organic non-bioavailable forms of selenium and the genetic manipulation of cellular selenium incorporation machinery indicated that selenoproteins were necessary for H-PGDS expression and 15d-PGJ2 production. Treatment of selenium-deficient macrophages with rosiglitazone, a peroxisome proliferator-activated receptor γ ligand, up-regulated H-PGDS. Furthermore, electrophoretic mobility shift assays indicated the presence of an active peroxisome proliferator-activated receptor-response element in murine Hpgds promoter suggesting a positive feedback mechanism of H-PGDS expression. Alternatively, the expression of nuclear factor-κB-dependent thromboxane synthase and microsomal PGE2 synthase was down-regulated by selenium. Using a Friend virus infection model of murine leukemia, the onset of leukemia was observed only in selenium-deficient and indomethacin-treated selenium-supplemented mice but not in the selenium-supplemented group or those treated with 15d-PGJ2. These results suggest the importance of selenium in the shunting of AA metabolism toward the production of PGD2 metabolites, which may have clinical implications.

The essential trace nutrient selenium plays a major role as a key component of the antioxidant machinery in all cell types. This ability of selenium is attributed, in part, to the co-translational incorporation of the amino acid selenocysteine (Sec) 3 into the active site of selenoproteins, many of which have important enzymatic functions (1). Proteins of the glutathione peroxidase (GPX) and thioredoxin reductase families are well characterized selenoenzymes that are involved in the regeneration of antioxidant systems and the maintenance of intracellular redox state and membrane integrity, as well as gene regulation by redox control of binding of transcription factors to DNA (2). Epidemiological studies suggest that inadequate selenium levels and associated reactive oxygen and nitrogen species production are linked to increased incidence of cardiovascular diseases, viral infections, including progression of HIV-AIDS and Alzheimer disease, infertility, and most notably cancer (2)(3)(4)(5). Supra-nutritional doses of selenium have demonstrable anti-carcinogenic effects in a variety of cancers through mechanisms such as perturbation of tumor cell metabolism, induction of apoptosis, and inhibition of angiogenesis (6). Also, supplementation of selenium has been shown to improve the health status of patients suffering from inflammatory conditions such as septic shock, autoimmune thyroiditis, pancreatitis, allergic asthma, and rheumatoid arthritis (2,(7)(8)(9). These anti-inflammatory actions can be partly explained by the antioxidant role of various selenoproteins, but the precise mechanisms in the context of specific cell types involved in inflammation need to be explored.
Macrophages are cells of the immune system that are central to the progression of a physiological, protective inflammatory response into a pathological, tissue-destructive inflammatory state. An imbalance between the production of reactive oxygen and nitrogen species and the scavenging anti-oxidant mechanisms forms the basis for such progression (10). In addition to the oxidative ("respiratory") burst reaction, classical macrophage activation is also characterized by the production of several mediators such as IL-1, IL-6, TNF␣, prostaglandin E 2 (PGE 2 ), and thromboxane A 2 (TXA 2 ) (11). PGE 2 , TXA 2 , PGD 2 , and PGD 2 downstream products, ⌬ 12 -PGJ 2 and 15d-PGJ 2 , are the major eicosanoids derived from the fatty acid arachidonic acid (AA) in macrophages (12), whereas PGI 2 is predominantly produced by vascular endothelial and smooth muscle cells that express high levels of PGI 2 synthase (PGIS) (13). The initial step of prostaglandin (PG) synthesis involves formation of PGH 2 from AA by the action of cyclooxygenase (COX)-1 and COX-2, which is subsequently acted upon by specific PG synthases, microsomal PGE synthase-1 (mPGES-1), thromboxane synthase (TXAS), and PGD synthase (PGDS), to form PGE 2 , TXA 2 , and PGD 2 , respectively (12). Studies have shown that during the resolution phase of inflammation, the AA metabolism shifts from the production of PGE 2 toward that of PGD 2 and its downstream double-dehydration product, 15d-PGJ 2 (13,14). This warrants further research into the regulation of the AA-COX pathway, which includes the various PG synthase enzymes. Oxidative stress in cells activates the transcription factor nuclear factor-B (NF-B). NF-B serves as a key transcription factor for mPGES-1 and TXAS, leading to the up-regulation of PGE 2 and TXA 2 , respectively (14,15). Our laboratory has previously shown that selenium supplementation of macrophages down-regulated NF-B with a corresponding increase in the activation of PPAR␥, thus affecting the production of PGE 2 (16 -18).
Lipocalin-type PGDS and hematopoietic PGDS (H-PGDS) are two isoforms that catalyze the conversion of PGH 2 to PGD 2 . While the former is expressed mainly in the CNS, the latter is found in immune cells like macrophages and mast cells, suggesting a role of H-PGDS in the regulation of inflammation (19,20). PGD 2 and 15d-PGJ 2 have been shown to possess anti-inflammatory properties, which are conferred via different mechanisms, including the activation of DP2 receptor, PPAR␥ activation, and covalent modification of cysteine thiols in target proteins such as IKK␤ and p65 of the NF-B signaling axis (20,21). However, not much is known about the molecular regulation of the glutathione (GSH)-dependent sigma-class glutathione S-transferase, H-PGDS. Studies from multiple laboratories, ours included, suggest that AA pathway is redox-sensitive (17,22,23). Selenium has been shown to modulate the expression of TXAS and prostacyclin synthase (PGIS) in endothelial cells (24). Furthermore, our laboratory has previously reported that selenium exerts its anti-inflammatory effects in activated macrophages via the enhanced production of 15d-PGJ 2 in a COX-1-dependent manner (25). This led us to the hypothesis that H-PGDS is regulated by the redox state of macrophages, which depends largely on their selenium status as well as the differential modulation of PPAR␥ and NF-B. Here, we demonstrate that selenium supplementation of macrophages differentially regulates the expression of mPGES-1, TXAS, and H-PGDS to favor the shunting of AA through the H-PGDS pathway where selenoproteins are indispensable. Furthermore, we present data that validate the shunting of PG metabolism using a mouse model of leukemia upon infection by Friend virus (FV), a retrovirus that infects hematopoietic progenitors in the bone marrow and spleen to cause development of leukemia (26). In this model, resistance of selenium-supplemented mice to FV infection was overcome with indomethacin treatment, although treatment with 15d-PGJ 2 , but not PGE 2 , completely rescued the selenium-supplemented mice despite inhibition of the AA pathway. Taken together, our data highlight the clinical significance of selenium-dependent shunting of AA toward PGD 2 metabolites.
Cell Culture and Animals-Murine macrophage-like cell line RAW264.7 (ATCC, Manassas, VA) was cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 5% fetal bovine serum (ATCC), 2 mM L-glutamine (Cellgro, Manassas, VA), penicillin-G (100 units/ml), and streptomycin (100 g/ml; Invitrogen). For routine culture, the cells were passaged every 3 days at a ratio of 1:10 as per the recommendations by ATCC. For experiments, the cells were seeded at a density of ϳ500,000/well of a 6-well plate and cultured for 2-4 days depending on the treatments. Selenite and Se-Met were dissolved in sterile water. p-XSC, ebselen, parthenolide, CAY10526, rosiglitazone, and 15d-PGJ 2 were dissolved in DMSO. The final concentration of DMSO in the cell culture medium was 0.1% v/v. Human embryonic kidney (HEK293T) cells were used for extracting nuclear proteins for the EMSA. They were cultured in DMEM containing 10% FBS, 2 mM L-glutamine, and penicillin/streptomycin. Primary murine macrophages (BMDMs) were prepared by extracting femoral bone marrow plugs from ϳ3-month-old C57BL/6 mice maintained on selenium-deficient (0.01 ppm selenite), selenium-adequate (0.1 ppm selenite), and selenium-supplemented (0.4 ppm selenite) diets for 12 weeks. These cells were then cultured in the same medium as described earlier with the addition of 20% L929 fibroblast-conditioned medium (as a source of granulocyte-macrophage colony-stimulating factor) for 1 week. Macrophages from selenium-deficient, selenium-adequate, and selenium-supplemented mice were cultured in defined media containing 7, 100, or 250 nM selenium (as selenite). For the FV-leukemia model, BALB/c mice (ϳ3 months old), maintained on selenium-deficient or selenium-supplemented diet (0.4 ppm selenite) for 12 weeks, were retro-orbitally injected with FV as described previously (27). These mice were dissected on day 15 post-infection, and signs of splenomegaly as well as WBC counts in the peripheral blood, characteristic of leukemia in this model (26,28), were examined. Hematological analysis was performed on an Advia 120 hematology autoanalyzer equipped with a veterinary software program. The COX inhibitor indomethacin was administered to the mice at a concentration of 0.00325% (w/v) in drinking water, as described previously (29), from 2 weeks prior to FV injection until 2 weeks post-infection, when the animals were euthanized. 15d-PGJ 2 or 11-deoxy-16,16-dimethyl-PGE 2 was exogenously administered at a concentration of 0.025 mg/kg/day (dissolved in PBS) intraperitoneally from day 7 to 14 post-infection to the respective groups. The special diets were formulated based on the recommended rodent diets from American Institute of Nutrition and purchased from Harlan-Teklad (Madison, WI).
Glutathione Peroxidase Activity Assay-Cells cultured under different concentrations of sodium selenite (0 -250 nM) were harvested and lysed with the mammalian protein extraction reagent (M-PER; Pierce) containing protease inhibitor mixture and 1 mM PMSF. Whole cell lysates were used for the GPX assay. The GPX activity was measured using H 2 O 2 and NADPH, and the activity was expressed as nanomoles of NADPH oxidized per min per mg of protein in a spectrophotometric assay as described earlier (30).
Arginase Activity Assay-Arginase activity, assessed by a colorimetric assay that detects urea production generated by arginase hydrolysis of L-Arg, has been described previously (31). The selenium-deficient and selenium-adequate RAW264.7 macrophages assayed were cultured as described earlier.
Absorbance at 560 nm was recorded on a Packard plate reader. A urea standard calibration curve (0 -1 mol; y ϭ 9E-05x ϩ 0.0007, R 2 ϭ 0.99) was used to calculate the arginase activity. Enzyme activity is expressed as micromoles of urea produced per mg of protein (31).
SDS-PAGE and Immunoblotting-Whole cell lysates were prepared from RAW264.7 cells or BMDMs using M-PER containing protease inhibitors, and protein concentration was estimated by BCA reagent. An equal amount of protein (ranging from 5 to 25 g) was electrophoresed on a 12.5% discontinuous SDS-polyacrylamide gel. Proteins were then transferred to a nitrocellulose membrane. The following primary antibodies were used to probe the membrane: anti-H-PGDS, anti-TXAS, anti-mPGES-1 (Cayman), anti-GPX1, anti-TR1 (Abcam, Cambridge, MA), and anti-GAPDH (Fitzgerald, Concord, MA). Appropriate secondary antibodies were used. Immunoreactive bands were visualized using the West Pico and/or West Femto chemiluminescence reagents obtained from Thermo Scientific (Rockford, IL). GAPDH was used as an internal control for normalization. Bands were evaluated by densitometry using the ImageJ software program (National Institutes of Health).
Real Time (Quantitative) and Semi-quantitative RT-PCR-RNA was isolated from RAW264.7 cells or BMDMs using the Isol-RNA lysis reagent (5 Prime, Gaithersburg, MD) as per its protocol. RNA purity and concentration were determined using UV spectroscopy. 2 g of DNase (New England Biolabs)treated RNA was used for reverse transcription reaction using High Capacity cDNA reverse transcriptase kit, per the manufacturer's instructions (Applied Biosystems, Foster City, CA). Prevalidated TaqMan probes for Hpgds, Mpges-1, Txas, and Pgis were purchased from Applied Biosystems (Foster City, CA). Gapdh probe was used as an internal control to normalize the data. Amplifications were performed using PerfeCTa quantitative PCR SuperMix master mix (Quanta Biosciences) in a 7300 real time PCR system (Applied Biosystems). ⌬Ct (Ct Gene Ϫ Ct Gapdh ) was calculated for each sample and used for analysis of transcript abundance with respect to the untreated negative control as described previously (32). For the semiquantitative RT-PCR, the sequences of the primers were as follows: mouse Hpgds sense 5Ј-ATGCCTAACTACAAACT-GCTT-3Ј and antisense 5Ј-CTAGAGTTTTGTCTGTGG-CCT-3Ј; mouse Gpx1 sense 5Ј-ACAGTCCACCGTGTATGC-CTTC-3Ј and antisense 5Ј-CTCTTCATTCTTGCCATTCTC-CTG-3Ј; and mouse ␤-actin sense 5Ј-TGGAATCCTGTG-GCATCCATGAAAC-3Ј and antisense 5Ј-TAAAACGCAG-CTCAGTAACAGTCCG-3Ј. PCR was carried out using 0.2 M of primers, 2.5 mM MgCl 2 , 0.2 mM of each deoxyribonucleotide triphosphate, 1.25 units of GoTaq DNA polymerase (Promega, Madison, WI), and 2.5 ng of template cDNA. After initial denaturation at 95°C for 2 min, PCR was continued with amplification cycles of 30 s at 95°C, 30 s at 56°C, and 1 min at 72°C, followed by elongation for 5 min at 72°C. The yield of PCR products was tested to be in the linear range, and optimal cycle number was chosen. The PCR products were separated by electrophoresis on a 1% agarose gel and visualized by UV transillumination. ␤-Actin was used as an internal control for normalization. Bands were evaluated by densitometry using the ImageJ program.
Transfections and Knockdowns-siRNA for Sec tRNA synthetase was obtained as ON-TARGET plus SMART pool (Thermo Scientific Dharmacon). 2 g of siRNA was transfected into 2 ϫ 10 6 RAW264.7 cells using Nucleofector Solution V according to the manufacturer's specifications (Amaxa Biosystems). All-Stars Negative Control siRNA (Qiagen) was used in equal amounts as a control. Selenite was added 12 h after transfection at a final concentration of 100 nM, and cells were harvested at 36 h after transfection for analysis by immunoblotting. The pU6-TetO4m4 vector containing shRNA target sequence, 5Ј-GACGTAGAGTTGGCATACC-3Ј (nucleotides 694 -712) for selenophosphate synthetase 2 (SPS2), was obtained from Dr. Dolph L. Hatfield, NCI, National Institutes of Health, Bethesda. The control vector was designated pU6Tet control, which consisted of the same construct minus the target sequence. For transfection, RAW264.7 macrophages were seeded into 6-well plates (1 ϫ 10 6 cells/well) without antibiotics. After 24 h, cells were transfected with either shSPS2 or empty vector using TransIT-LT1 (Mirus Bio) according to the manufacturer's specifications. The constructs were used at a final concentration of 2.5 g/well. Forty eight hours after transfection, cells were left in the media with or without 250 nM selenite for 48 h followed by 12 h of 1 g/ml LPS stimulation. Cells and media supernatants were collected for 15d-PGJ 2 quantitation, and the corresponding cell lysates were used for Western immunoblotting analyses. SPS2 knockdown stable RAW264.7 cells were also prepared by transfecting the shSPS2 or the empty vector as described above. Resistant clones were selected in 100 g/ml hygromycin and then expanded, followed by culturing them in a selenite-containing media to examine the knockdown of selenoproteins. Western blot analysis of SPS-2 confirmed ϳ80% knockdown. Approximately 60% confluent HEK293T cells in a 60-mm culture dish were transfected with 6 g of pCDNA3.1 containing human PPAR␥ full-length cDNA (provided by Dr. Jack Vanden Heuvel, Pennsylvania State University) using TransIT-293 (Mirus Bio) transfection reagent. Rosiglitazone was added 24 h later to a final concentration of 2 M for the expressed PPAR␥ to translocate into the nucleus. After 24 h, cells were harvested to prepare nuclear extracts.
PG Quantitation-The concentrations of PGE 2 and TXB 2 in culture media supernatants of BMDMs and RAW264.7 cells treated with selenium as indicated and treated with 1 g/ml LPS were determined by enzyme immunoassay according to the manufacturer's instructions (Cayman) and normalized to total cellular protein. 15d-PGJ 2 was quantitated from supernatants from RAW264.7 cells transiently transfected with shSPS2 by using enzyme immunoassay kit from Assay Designs (Ann Arbor, MI). In most cases, the values were normalized to total cell protein.
LC-MS Analysis of 15d-PGJ 2 and ⌬ 12 -PGJ 2 Production-The RAW264.7 or BMDM cell culture supernatants were acidified with HCl (1 N) to pH 3.0 and extracted with ethyl acetate. The compounds 15d-PGJ 2 and ⌬ 12 -PGJ 2 were analyzed using an LC-MS/MS system comprising of Shimadzu LC20AD UFLC pumps and API2000 triple quadruple mass spectrometer set to negative mode with a temperature of 200°C for the confirmation of molecular ions. The solvent system used was methanol/ H 2 O (70:30) with 0.1% acetic acid at a flow rate of 0.15 ml/min. The MS analysis was performed on API2000 set to scan mode (m/z 100 -350) for authentic standards, and molecular ions m/z 315.6 (M Ϫ H ϩ ) for 15d-PGJ 2 and 333.5 (M Ϫ H ϩ ) for ⌬ 12 -PGJ 2 , respectively, were used for quantitation. As an internal control to calculate extraction efficiency, 200 ng of deuterated 15d-PGJ 2 (d4) (Cayman Chemicals) was used, which showed a 70% extraction efficiency. Standard calibration curves for 15d-PGJ 2 and ⌬ 12 -PGJ 2 were set up for the quantitation, and calculations were performed based on the following equations: y ϭ 76.831x Ϫ 608.42; r 2 ϭ 0.9927 (for 15d-PGJ 2 ) and y ϭ 7.2516x ϩ 172.34; r 2 ϭ 0.9871 (for ⌬ 12 -PGJ 2 ). As described earlier, the values were normalized to total cell protein.
Statistical Analysis-The results are expressed as means Ϯ S.E. The differences between groups were analyzed by Student's t test using GraphPad Prism. The criterion for statistical significance was p Ͻ 0.05. RAW264.7 Macrophages-To examine the regulation of H-PGDS by selenium, RAW264.7 macrophages cultured for 3 days in various concentrations of exogenously added selenium (0 -500 nM selenium as selenite) were used. Analysis of the cell lysates displayed a dose-dependent increase in the cytosolic GPX enzyme activity (Fig. 1A). Although the level of GPX was very low in selenium-deficient conditions, 100 nM selenium induced an ϳ10-fold increase in the GPX activity and protein expression (Fig. 1, A and B). Using this model, we examined the ability of selenium to up-regulate expression of H-PGDS. As seen with GPX activity, the expression of H-PGDS also increased as a function of selenium concentration, with saturation at 150 -250 nM selenium. We observed an ϳ3-fold increase in protein levels of H-PGDS at 250 nM (Fig. 1B). In concert with the increased levels of H-PGDS, downstream metabolites of PGD 2 , ⌬ 12 -PGJ 2 , and 15d-PGJ 2 also showed a similar dose-dependent increase with selenite in the cell culture supernatants using quantitative LC/MS-based assays (Fig. 1C). At all the doses of selenium tested, the concentration of ⌬ 12 -PGJ 2 was much higher than its dehydrated metabolite 15d-PGJ 2 .

Selenium Up-regulates H-PGDS Expression and Its Downstream Metabolites in
Selenite Differentially Regulates the Expression of PG Synthases in RAW264.7 Cells and BMDMs-To investigate the role of selenium in the shunting of the AA-COX pathway, the expression of two other additional PG synthases, mPGES-1 and TXAS, which are highly expressed in macrophages, were examined by immunoblotting and real time RT-PCR. With an increase in selenium, the expression of mPGES-1 and TXAS showed a dose-dependent decrease at both protein and transcript levels. However, transcript levels of Hpgds increased as a function of selenium, in agreement with the protein level as described earlier (Fig. 2, A and B). Analysis of the expression levels of Pgis showed an interesting biphasic regulation in that an initial increase in the transcript was seen with 50 and 100 nM selenium (Fig. 2B). However, further addition of selenium (Ͼ100 nM) decreased the levels of Pgis. Analysis of protein expression of PG synthases in primary BMDMs isolated from the bone marrow of mice on diets containing different selenium concentrations showed a dose-dependent increase in H-PGDS, whereas mPGES-1 and TXAS expression was decreased at 100 and 500 nM of selenium relative to selenium-deficient cells (Fig. 2C).
Different Forms of Selenium Have Variable Effects on Expression of PG Synthases in RAW264.7 Cells and BMDMs-To further understand the selenium-dependent regulation of the AA-COX pathway, the effects of four commonly used forms of selenium were compared. RAW264.7 cells were incubated with 0, 100, and 500 nM of selenium in the form of sodium selenite, MSA, p-XSC or Se-Met for 3 days and analyzed for protein and transcripts of H-PGDS and other PG synthases. GPX1 levels were also examined as a marker of selenoprotein status of these cells. Fig. 3, A and B, shows that MSA up-regulated Hpgds in a manner comparable with sodium selenite treatment, but p-XSC and Se-Met did not lead to a significant change in H-PGDS or GPX1 from the base-line levels. Conversely, expression of Mpges-1 and Txas was down-regulated with MSA but not Se-Met and p-XSC. Real time RT-PCR analyses on BMDMs (from selenium-deficient mice) treated with different forms of selenium ex vivo showed similar patterns of expression of PG synthases (Fig. 3C), an exception being the down-regulation of Txas by Se-Met. Quantification of the levels of PGE 2 and TXB 2 in RAW264.7 and BMDM cell culture supernatants by ELISA confirmed the results obtained from the expression analyses of PG synthases (Fig. 3, D and E). Analyses of the BMDM supernatants by LC/MS for ⌬ 12 -PGJ 2 and 15d-PGJ 2 revealed significantly high levels with the 500 nM selenite treatment and minor increases within the other groups as compared with the selenium-deficient samples (Fig. 3F). These results suggest that increased bioavailability of selenium, perhaps in the form of selenoproteins, is a critical determinant of selenium-dependent regulation of PG synthases in macrophages.

Selenoprotein Synthesis Is Critical to Regulate the Expression of H-PGDS by Selenium-
To examine if incorporation of selenium into selenoproteins was essential for the up-regulation of H-PGDS and subsequent formation of 15d-PGJ 2 , a genetic knockdown approach was used. Selenium-deficient RAW264.7 macrophages were transfected with either shSPS2 or control constructs and then maintained under selenium-deficient and selenium-supplemented (250 nM) conditions for 2 days followed by 1 g/ml LPS stimulation for 12 h. The cell lysates were used for the analysis of the expression of SPS2, GPX1, and TR1 by immunoblotting. Although the expression of SPS2 (data not shown), GPX1, and TR1 was significantly knocked down in shSPS2-transfected cells despite the presence of selenium, there was no change in the expression of these proteins in cells transfected with the vector control construct (Fig. 4A). To confirm that SPS2 knockdown cells cannot produce 15d-PGJ 2 due to the lack of selenoproteins, the cell supernatants were utilized for the analysis of 15d-PGJ 2 production. The production of 15d-PGJ 2 was almost completely inhibited in SPS2 knockdown cells even after supplementing with 250 nM selenium. In contrast, we observed a significant increase in the production of 15d-PGJ 2 in those cells that were transfected with control constructs followed by selenite treatment (Fig. 4B). Evaluation of H-PGDS expression using the shSPS2 stably transfected macrophages that were cultured with 100 nM selenium showed a clear down-regulation of GPX1 as well as H-PGDS only in shSPS2 knockdown cells but not in the vector control (Fig. 4C). Furthermore, we genetically manipulated the expression of Sec -tRNA synthetase, an enzyme that is critical in the pathway of Sec incorporation into selenoproteins (33). Western immunoblotting of cells that were subjected to siRNA-mediated knockdown of Sec tRNA synthetase clearly showed a substantial downregulation of H-PGDS and GPX1 (Fig. 4D). Interestingly, treatment of Se-Met in presence of r-METase, an L-Met ␣,␥lyase that releases selenium for incorporation into selenoproteins, led to the up-regulation of H-PGDS expression (Fig. 4E). To understand if the activity of GPX, one of the most abundantly expressed selenoproteins, was pivotal to up-regulate H-PGDS, ebselen, a small molecular mimetic of GPX1 (34), was used. RAW264.7 cells treated with different concentrations of ebselen showed a dose-dependent increase in levels of Hpgds transcripts in the absence of any increase in GPX1 (Fig. 4F). Taken together, these results provide further evidence in support of the important role of selenoproteins in the up-regulation of H-PGDS and subsequent shunting of the AA-COX pathway in macrophages.
PPAR␥-dependent Transcription of H-PGDS-We speculated the presence of a positive-feedback regulation of H-PGDS by the endogenous PPAR␥ ligand, 15d-PGJ 2 . Examination of the murine Hpgds promoter for the presence of PPAR-response elements (PPREs) using MatInspector (Genomatix Software Gmbh, Munich, Germany) resulted in at least four putative binding sites at Ϫ1250, Ϫ3401, Ϫ4478, and Ϫ4896 bp for PPAR␥ found within 5 kb upstream of the transcription start site of Hpgds, which have a matrix similarity score of Ͼ0.75 when compared with a cPPRE (Fig. 5A). To examine the binding of these PPREs to PPAR␥ within the context of the promoter, we analyzed the binding of oligonucleotides containing each of these PPREs to HEK293T-expressed PPAR␥. Gel shift analysis clearly showed that PPRE1 (Ϫ1250) interacted strongly with PPAR␥, somewhat similar to the cPPRE, whereas PPRE3 (Ϫ4478) was relatively ineffective, and PPRE2 (Ϫ3401) and PPRE4 (Ϫ4896) showed minimal binding (Fig. 5B). We confirmed the specificity of PPAR␥ binding to PPRE1 by competing 32 P-labeled PPRE1 oligonucleotide with varying amounts of unlabeled cPPRE oligonucleotide as well as by displaying a supershift using anti-PPAR␥ antibody (Fig. 5C). Having located functionally active PPREs (Ϫ1250 and Ϫ4896) in the murine Hpgds promoter, we studied the regulation of H-PGDS expression upon treatment of RAW264.7 macrophages with rosiglitazone, a bona fide agonist of PPAR␥. Rosiglitazone significantly increased H-PGDS protein expression in selenium-deficient cells (Fig. 5D). These studies indicate that PPAR␥ plays a critical role in the regulation of H-PGDS expression, which is facilitated by selenium.
Inhibition of H-PGDS Blocks Selenium-mediated Up-regulation of PPAR␥-dependent Arginase-1 Production-Having previously shown that there is increased nuclear translocation of PPAR␥ in the presence of selenium in macrophages (25), and knowing that macrophage alternative activation marker arginase-1 (Arg-I) is induced by PPAR␥ (35) and increases in CyPGs in selenium-treated cells, we studied the effect of selenium on the activation of arginase-1 and the role of H-PGDS in this process. Treatment of RAW264.7 macrophages with 100 nM selenium increased the arginase-1 activity up to ϳ7-fold compared with the selenium-deficient cells in the presence of IL-4, which is a Th2 cytokine essential for alternative activation. A similar increase in the transcript levels of Arg-I was also observed (data not shown). Interestingly, pretreatment of cells with an H-PGDS inhibitor, HQL-79, completely blocked the selenium-mediated increase in arginase-1 (Fig. 6). Treatment of selenium-supplemented cells with HQL-79 also inhibited the production of ⌬ 12 -PGJ 2 and 15d-PGJ 2 (data not shown).  AUGUST 5, 2011 • VOLUME 286 • NUMBER 31

JOURNAL OF BIOLOGICAL CHEMISTRY 27477
Control of NF-B Activation Shunts the AA Pathway toward PGJ 2 Production-Given that NF-B is known to regulate the expression of mPGES-1 and TXAS (14,15), we examined whether inhibition of NF-B in selenium-deficient cells provides an alternative route of metabolism of PGH 2 , possibly through H-PGDS, which provides sufficient 15d-PGJ 2 to upregulate H-PGDS. To test this potential feedback activation, RAW264.7 macrophages were treated with 1 M parthenolide (PTN) for 1 h before stimulation with 1 g/ml LPS. Inhibition of NF-B with PTN up-regulated H-PGDS expression (transcripts and protein) under selenium-deficient conditions. An increase in H-PGDS was accompanied by a decrease in TXAS and mPGES-1 expression (Fig. 7, A and B). Similarly, inhibition of mPGES-1 by CAY10526 in these cells, followed by LPS stimulation, resulted in an increase in H-PGDS expression (Fig. 7, A and B) as well as increased ⌬ 12 -PGJ 2 and 15d-PGJ 2 levels in the supernatants, when analyzed by LC-MS (Fig. 7C).
Selenium Supplementation Prevents Development of Disease in FV Murine Leukemia Model-To demonstrate the significance of selenium in an in vivo disease model with regard to the AA pathway, we used the FV murine leukemia model, where the infection of mice with the retrovirus leads to the expansion of a clone of infected hematopoietic precursors in the spleen and bone marrow that results in the development of acute leukemia (26). The disease is characterized by splenomegaly within 2 weeks of FV infection, accompanied by a significant rise in the WBC count (28). Selenium-deficient FV-infected BALB/c mice showed pathological changes demonstrated by the ϳ12-fold increase in spleen size (Fig. 8, A and B) and ϳ4-fold increase in the WBC counts (Fig. 8C), although FVinfected mice maintained on selenium-supplemented diets showed no signs of disease. Interestingly, administration of indomethacin to selenium-supplemented mice caused splenomegaly with an increase in WBC counts upon FV infection as in the selenium-deficient mice (Fig. 8, D-F). Daily intraperitoneal administration of 15d-PGJ 2 at 0.025 mg/kg to a group of indomethacin-treated selenium-supplemented mice rescued them from the FV leukemia, although administration of a stable PGE 2 analog failed to rescue these mice (Fig. 8, D-F). The dose of 15-PGJ 2 used in the above experiment was based on a doseresponse study to determine the minimal dose required to com-  pletely alleviate the signs of the disease (data not shown). Taken together, these studies indicate the critical role of seleniummediated shunting of the AA pathway in an in vivo pathogenesis model.

DISCUSSION
Epidemiological evidence suggests that the benefits of the essential micronutrient selenium in optimal health are, in part, contributed by the ability of selenium to alleviate inflammatory signaling pathways (2,5). Selenium might impart these antiinflammatory properties via different selenoproteins, but the mechanisms have not been completely understood. These selenoproteins form an important component of the cellular redox regulatory machinery, especially in oxidative stressprone macrophages. Inhibition of the activation of the NF-B signaling axis and its effect on the down-regulation of its proinflammatory gene products such as COX-2, mPGES-1, and TXAS are well known. We have earlier shown that selenium can inhibit NF-B activation in macrophages (16 -18). Given the fact that macrophages, among other immune cells, have the enzymatic machinery to metabolize AA to all PG classes, including TXA 2 , and that PGD 2 metabolites produced by these  A and B show the spleens and spleen weights, respectively. C shows the WBC counts on blood samples from each group. D and E show spleens and spleen weights, respectively, from selenium-supplemented indomethacin (Indometh) (0.00325% w/v in drinking water)-treated BALB/c mice. 15d-PGJ 2 or PGE 2 analogs were administered intraperitoneally at a concentration of 0.025 mg/kg/day from day 7 to day 14 post-infection in respective groups. F shows WBC counts on blood samples from each group. n ϭ 4 mice in each group. *, p Ͻ 0.05. cells have the ability to modulate NF-B and PPAR␥ pathways (19 -21), we explored the possibility of selenium regulating the production of different PGs at the transcription level. In this study, we report that bioavailability of selenium for incorporation into selenoproteins is essential to up-regulate the expression of H-PGDS and down-regulate the expression of mPGES-1 and TXAS. The resultant increase in H-PGDS shunts the metabolism of the AA-derived COX product, PGH 2 , from proinflammatory PGE 2 and TXA 2 toward the anti-inflammatory and pro-resolution product 15d-PGJ 2 . As a proof-of-concept of the translatability of these studies, we have utilized an FVinfection leukemia model. Our study demonstrates the protective effect of selenium to be inhibited by indomethacin treatment of mice, although subsequent treatment of such mice with 15d-PGJ 2 , but not PGE 2 , completely inhibited the disease. These studies corroborate the current ex vivo and previous studies of the in vivo production of 15d-PGJ 2 in selenium-supplemented mice (25) as a critical mechanism underlying the anti-leukemic effect of selenium. However, the exact mechanism and targets of selenium or endogenous 15d-PGJ 2 remain to be characterized.
The dose-dependent increase in the activity and expression of GPX1 with sodium selenite reinforces the fact that GPX1 is a reliable marker of cellular selenium status in macrophages. The saturation of GPX1 activity and expression at 100 nM selenium in the form of selenite added to selenium-deficient cells demonstrate the sensitivity of the selenoprotein synthesis machinery to bioavailable selenium. Therefore, we subjected seleniumdeficient primary BMDMs and RAW264.7 cells to different doses (0 -500 nM) of selenite to examine if there was an effect on the transcription of H-PGDS, which is the rate-limiting step for the formation of PGD 2 and its stable metabolites, ⌬ 12 -PGJ 2 and 15d-PGJ 2 . The dose-dependent up-regulation of H-PGDS along with the increased production of its downstream products by selenite, in both primary and immortalized macrophages, demonstrate that the cellular selenium status plays a pivotal role in regulating the expression of proteins outside the selenoproteome. Higher levels of ⌬ 12 -PGJ 2 as compared with 15d-PGJ 2 in the cell culture supernatant of primary cells than those in the immortalized cells are consistent with those reported for eicosanoids in general by Rouzer et al. (36). Also, the increased accumulation of ⌬ 12 -PGJ 2 than its dehydrated product (15d-PGJ 2 ) is also in agreement with the reports on the thermodynamically unfavorable conversion of ⌬ 12 -PGJ 2 to 15d-PGJ 2 in the presence of proteins (37,38). Conversely, the dose-dependent down-regulation of mPGES-1 and TXAS and their pro-inflammatory products PGE 2 and TXA 2 at similar concentrations of selenite indicate a favorable skewing of AA-COX pathway metabolites. This regulation by selenium could be a direct consequence of the change in the redox state of the cell affecting the transcription of many diverse redox-sensitive proteins or an indirect consequence of the activation of signaling pathway(s), which eventually regulates H-PGDS expression. Along these lines, treatment of selenium-supplemented cells with buthionine-sulfoximine (an inhibitor of GSH synthesis) completely abrogated the selenium-dependent increase in 15d-PGJ 2 (data not shown). Taken together, our data argue that selenium in the form of selenoproteins appears to be crucial for this effect. Evidence that selenium can modulate PGIS expression in endothelial cells (24) prompted us to look at the regulation of this enzyme in our macrophage model. We found that PGIS is expressed in insignificant quantities, which is consistent with the fact that PGIS is predominantly expressed in endothelial cells and smooth muscle cells (13).
Interestingly, our studies demonstrate that selenium in different forms variably affects the expression of PG synthases. In cell culture models, sodium selenite and MSA are known to make selenium easily bioavailable for the formation of Sec, which is incorporated into selenoproteins during translation (3). However, Se-Met and p-XSC do not release selenium for selenoprotein synthesis as reflected by their inability to increase GPX1 expression above the base-line levels (Fig. 3A). The expression of H-PGDS follows the same pattern as GPX1 for all of these forms of selenium, which suggests that selenoproteins appear to be involved in the up-regulation of H-PGDS expression. Similar observations obtained from primary BMDM at the level of transcripts and end products suggest that the regulation of PG synthases by selenium, in the form of selenoproteins, is not just a feature of immortalized cell lines such as the RAW264.7 macrophage-like cell line. Apart from using genetic methods to knock down the expression of selenoprotein, we have taken the absence of ␥-lyase in our in vitro culture system to our advantage to provide support for the role of selenoproteins in AA metabolism. This may not be the case at the organismal level where ␥-lyase is expressed mostly in the kidney to metabolize these compounds to contribute, in part, to the anticarcinogenic properties of such compounds as p-XSC (39,40). Alternatively, it is also possible that these compounds may target signaling pathways even without involving selenoproteins to impart their beneficial actions. Compounds like Se-Met require an enzymatic reaction involving a ␥-lyase to release selenium. Thus, when the macrophages were treated with Se-Met and r-METase, there was a dose-dependent increase in H-PGDS, which continues to support the involvement of selenoproteins to play a key role in the expression of H-PGDS. Furthermore, the genetic knockdown of two critical enzymes in the selenoprotein incorporation pathway, SPS-2 and Sec tRNA synthetase, also led to the down-regulation of H-PGDS even in the presence of selenium. These results make a compelling case to identify the selenoprotein(s) that are crucial for the regulation of H-PGDS expression and subsequent production of the potent anti-inflammatory mediator 15d-PGJ 2 . Based on our studies with NF-B inhibitors, we speculate that changes in the redox status of cells, as a result of the modulation of selenoprotein expression, could likely be the underlying mechanism. The ability of GPX-mimetic ebselen to up-regulate H-PGDS in selenium-deficient macrophages also indicates that redox modulatory pathways are likely to contribute to the shunting of AA. These are very interesting aspects that justify exploration in the future.
The regulation of H-PGDS at the transcriptional level and the already documented finding from our laboratory that selenium supplementation of macrophages leads to an increase in activation of PPAR␥ (25) mediated by the increased levels of an endogenous PPAR␥ ligand, 15d-PGJ 2 , led us to examine the PPAR␥-dependent regulation of the murine H-PGDS gene.
Our results from the PPAR␥ gel shift assays clearly indicate that PPAR␥ binds to the promoter of Hpgds at one or more of the four PPREs, with the PPRE at Ϫ1250 bp (PPRE1) being the strongest. Likewise, the up-regulation of H-PGDS by the prototypic PPAR␥ ligand rosiglitazone, in the absence of selenium, indicates that H-PGDS is indeed a PPAR␥-dependent gene.
These findings also open an intriguing possibility of feedback regulation of H-PGDS by 15d-PGJ 2 in the selenium-supplemented macrophages. It is possible that in addition to PPAR␥, PPAR␤ could also play a critical role in the up-regulation of H-PGDS because PPAR␤ is highly expressed in macrophages and is known to possess a variety of anti-inflammatory properties (41,42). However, preliminary promoter analyses for PPAR␤-binding sites and the inability of PPAR␤-specific synthetic agonists to activate H-PGDS expression suggest that the binding activity is perhaps restricted to PPAR␥ (data not shown).
Besides being a PPAR␥ ligand, 15d-PGJ 2 has been shown to inhibit the activation of pro-inflammatory genes by covalently binding specific cysteine residues in IKK␤, a pivotal enzyme in the canonical pathway of NF-B activation (43). Thus, in selenium-supplemented macrophages, the up-regulation of H-PGDS leads to the enhanced production of 15d-PGJ 2 , which not only acts in a positive feedback loop to up-regulate H-PGDS transcription in a PPAR␥-dependent manner, but also inhibits NF-B target genes, including mPGES-1 and TXAS enzymes, in LPS-stimulated selenium-supplemented macrophages. To provide more evidence for the shunting of the PG synthesis pathway, we used parthenolide (PTN), a well known NF-B inhibitor, to inhibit mPGES-1 and TXAS expression to create an increase in the availability of PGH 2 for H-PGDS. Alternatively, inhibition of mPGES-1 by CAY10526 also mimicked the effect of PTN with respect to increased H-PGDS expression and subsequent ⌬ 12 -PGJ 2 and 15d-PGJ 2 production. Taken together, the subsequent up-regulation of H-PGDS under selenium-deficient conditions strongly supports the possibility of a feed-forward loop involving H-PGDS 3 15d-PGJ 2 3 PPAR␥ 3 H-PGDS (Fig. 9).
In summary, our studies demonstrate a critical role for selenium in anti-inflammation that also has a bearing on the protection from malignant transformation of WBCs. This is particularly relevant because macrophages are implicated in both the initiation and resolution of inflammation. Thus, it is important to understand how exactly their functions are so tightly regulated. Although the initial phases of inflammation involve increases in levels of pro-inflammatory mediators like PGE 2 and TXA 2 , a switch toward the pro-resolving and antiinflammatory mediators like 15d-PGJ 2 during the latter stages suggests the presence of a critical regulator being involved. As seen with the increased activity of arginase in macrophages treated with selenium, shunting of the AA pathway, particularly in macrophages, may have many implications, the most notable being the switch from classically activated "M1" macrophage to alternatively activated "M2" macrophage phenotypes that have wound-healing and resolving properties in addition to affecting the progression of FV leukemia. Such studies delineating the underlying mechanisms centered on the common theme of "lipid class" switching by micronutrient selenium are being carried out in the laboratory and will be reported in the near future.