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Regulation of Proinflammatory Gene Expression by Selenium K. Sandeep Prabhu, Ph.D. Center for Molecular Toxicology & Carcinogenesis and Center for Molecular Immunology & Infectious Disease The Huck Institutes of the Life Sciences Department of Veterinary and Biomedical Sciences The Pennsylvania State University, University Park, PA www.vetsci.psu.edu • Physiology and biochemistry of Selenium (Se) • Effect of Se on oxidative stress-induced proinflammatory gene expression • Redox regulation of NF-kB: Endogenous inhibitor of NF-kB in Se-supplemented mammalian cells • Regulation of the prostaglandin pathway Chemistry of Selenium Discovered by J. J. Berzelius (1817) Third member of Group VI A Belongs to Oxygen family Electronic configuration: 1s22s22p63s13p63d104s24p4 Valence Numbers:-2, +2, +4 and +6 http://environmentalchemistry.com/yogi/periodic/Se.html#Overview Dietary Sources of Se • • • • • • • • Brazil nuts, dried, unblanched, 1 oz: 840 mcg Tuna, canned in oil, drained, 3 1/2 oz: 78 mcg Noodles, enriched, boiled, 1 c: 35 mcg Turkey, breast, oven roasted, 3 1/2 oz: 31mcg Chicken, meat only, 1/2 breast: 24 mcg Bread, enriched, whole wheat, 2 slices: 20 mcg Oatmeal, 1 c cooked: 16 mcg Cottage cheese, lowfat 2%, 1/2 c: 11 mcg Tissue Distribution Human Rat __________________________________________ Tissue/ Se Se organ (mg/kg) (mg/kg) __________________________________________ Muscle 0.24 0.12 Skeleton 0.42 0.15 Liver 0.54 0.78 RBC 0.29 0.54 Plasma 0.13 0.52 Fatty Tissue 0.04 0.04 Testes 0.30 0.90 __________________________________________ http://www.selevel.org/default.shtml Source: Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids (2000) IOM, Natl Academies Selenium-Related Problems in Human Health Hypothyroidism Colon Cancer Cardiovascular Diseases Arthritis Prostate Cancer HIV HIV Cytokine levels of IL-2, TNF-a, IL-8 decreased by Se-supplementation Se impacts T-lymphocyte proliferation and differentiation Mastitis Selenium deficiency causes increased neutrophil adherence in bovine coliform mastitis Bovine mammary endothelial cells cultured in Se-deficient exhibit increased expression of E-Selectin, ICAM-1, cyclooxygenase-2, and lipoxygenases Selenium supplementation in the bovine improved the outcome of coliform mastitis Cardiomyopathy: Keshan Disease : endemic dilated cardiomyopathy with multiple focal necrosis, cell infiltration; various stages of fibrosis, myopathy, necrotic myopathy (white-muscle disease) Selenium deficiency may permit the mutation of normally benign Coxsackie viruses into cardiotoxic strains. Is Se-deficiency Common? •Geochemical Factors-Soil Se Content •HIV Patients •Coronary Atherosclerosis •Breast cancer patients •Cigarette Smokers •Statin Users Selenium Toxicity-Selenosis • Gastrointestinal upsets, hair loss, white blotchy nails, and mild nerve damage • The rare cases of selenium toxicity in the US have been associated with industrial accidents and a manufacturing error that led to an excessively high dose of selenium in a supplement • The Institute of Medicine has set a tolerable upper intake level for selenium at 400 micrograms per day for adults to prevent the risk of developing selenosis Antioxidant Cycling Metabolism of Inorganic and Organic Forms of Selenium Plants Se-Proteins SeCys SeMet LYAS E S Selenate GSH Selenite MeSeCys [H] +Me H2Se Selenide -Me CH3SeH Methyl Selenol TMSe Selenoproteins Excretion Mechanism of Sec insertion in eukaryotes Sec = Selenocys Animal Sec-containing proteins. All currently known selenoproteins are listed (left). The relative sizes of selenoproteins (empty boxes) and the locations of Sec (red box) and an αhelix immediately downstream of Sec (green box) in the selenoprotein sequences are indicated (right). Kryukov et al (2003) Science, 300(5624):1439-43 Selenoenzymes Mammalian enzymes— Glutathione peroxidases (GPX) GPX1 Classical glutathione peroxidase (GSH-Px) GPX2 Gastrointestinal glutathione peroxidase (GPX-GI) GPX3 Plasma glutathione peroxidase (Plasma GPX) GPX4 Phospholipid hydroperoxide GPX (PHGPX) GPX5 Androgen-regulated epididymal secretory protein GPX6 Odorant-metabolizing protein Thioredoxin reductases (TrxR1-4) Deiodonases (DI) DI1 Iodothyronine 5’-deiodonase-1 (type 1 DI) DI2 Iodothyronine 5’-deiodonase-1 (type 2 DI) DI3 Iodothyronine 5’-deiodonase-1 (type 3 DI) Sel-P Plasma selenoprotein P Assessment of Adequacy of Se Status: [Se] mM Prevention of Keshan disease Optimal activity of IDs Maximization of plasma GPX, SePP Protection against some cancers Source: Thomson CD (2004) Eu J Clin. Nutr. 58: 391-402 >0.25 >0.82 >1.0-1.2 >1.5 Selenoproteins •Selenium dependent Glutathione Peroxidase 2GSH NADP+ ROOH Se-GPX NADPH GSSG ROH + H2O •Thioredoxin reductase (TrxR) Trx-(SH)2 NADP+ TrxR NADPH Trx-S2 ROOH ROH + H2O PMA Receptor ligation NADPH OXIDASE (NOX) Fibroblasts T- & B-cells Endothelial cells Cytosolic SODs H 2 O2 Schematic of the VEGF-Mn-SOD signaling axis ROS-Generating Enzymes • • • • • NADPH Oxidase Cyclooxygenases (COX) Lipid intermediates Lipoxygenases (LOXs) SODs Fe & Cu-dependent enzymes (Fenton Chemistry) Reactive Oxygen Species (ROS) NADPH Oxidase eO2 O2 oxygen hydrogen peroxide NO. NOS e- H+ ONOOperoxynitrite Citrulline e- e- H2O hydroxyl radical ONOOH TrxR/Trx GPx H2O + O2 O2 O2 OH + HO - peroxonitrous acid Flavin-containing enzymes .- SOD O2 . H2O2 superoxide Arginine O2 e- .- H2O2 Catalase . Lipid-peroxidation OH .-Mn-SOD H2O2 Damage to DNA/Protein ROS/RNS Physiological functions • Defense against infection • ROS can act as second messengers in signal transduction pathways • Protein phosphorylation and transcription factor binding are influenced by cellular oxidant/antioxidant balance Cause of oxidative damage • DNA damage • Lipid peroxidation • Protein modification Increased ROS production results from an oxidant-antioxidant imbalance Antioxidants Selenium Vitamins C/E Glutathione (GSH) Lipoic acid N-Acetyl Cys (NAC) ROS H2O2, OH-, O2.-, NO. Fatty acid hydroperoxides Prostaglandins Leukotrienes Cancer AIDS Arthritis Atherosclerosis Alzheimer's Aging Diabetes Mastitis (bovine) Intracellular ROS Sensors • Phosphorylation of kinases • S-Thiolation of Cys residues in kinases and phosphatases • Nitrosylation • Michael adducts with Prostaglandins, lipid peroxidation products of kinases Regulation of Cellular ROS levels by Selenoenzymes Se-dependent GPX and TrxR activities are dependent on the Se status in Rats Filled bars: 0.2 mg Se/g diet as Na2SeO3; Open bars: 0.008 mg Se/g diet; T= 28 days RAW264.7/Murine Bone Marrow-derived Macrophage Model Mice (3 wk) 3 mo Se-supplemented diet (0.4 ppm) Se-deficient diet (0.01 ppm) RAW264.7/BMDM in DMEM with 2 mM Gln,100 Units/ml Penicillin-G, 100 mg/ml Streptomycin, 5 % FBS (defined) + M-CSF (50 ng/ml) No Se added + 0.25 - 2 nmol/ml Sodium Selenite Repletion Se-deficient (Se-) Depletion Se-supplemented (Se+) GPX1 in Se-deficient and Se-supplemented cells RAW264.7 BMDM Se-Deficiency Increases Total ROS Pathways of NF-kB Activation NF-kB Pathway Cell survival Pro-inflammatory cytokines Proinflammatory enzymes Adhesion molecules Activation of PSA Tumor initiation, promotion, and progression Inactivation of JNK Inhibition of NF-kB in cancer cells converts inflammation-induced tumor growth to tumor regression Luo, J.-L. et al. J. Clin. Invest. 2005;115:2625-2632 Copyright ©2005 American Society for Clinical Investigation CAPE: Inhibits the activation of NF-kB Caffeic acid phenethyl ester •Isolated from propolis of honeybee hives •Potent and specific inhibitor of NF-kB activation induced by different agents •Mechanism is still unknown Natarajan et al (1996) PNAS (USA) 93, 9090-9095 Guggulsterone inhibits NF-kB activation Plant sterol from gum resin of Commiphora mukul GS suppressed DNA binding of NF-kB induced by TNFa, PMA, Okadaic acid, cig. Smoke Mechanism: Inhibition of IKK activity? Shishodia S and Aggarwal BB (2004) J. Biol. Chem. 279, 47148-47158 Shishodia and Aggarwal, 2005 Activation of NF-kB in Se-deficiency Selenium-supplementation can inhibit the activation of NF-kB in macrophages Luc Luciferase reporter vector kB kB kB kB kB Zamamiri-Davis et al (2001) Free Radic. Biol. Med. Se-deficiency increases nuclear translocation of p65 and p50 proteins in HepG2 cells Se-deficient LPS 0 2 4 6 Se-supplemented 8 12 0 2 4 6 8 12 (h) IB:p65 IB: p50 Se-Deficiency Exacerbates TNFa Production in BMDM COX Isoforms Two known isoforms: COX-1 and COX-2 Share 60% sequence homology, aspirin acetylation and glycosylation sites Differ significantly at cellular, genetic, pathological and pharmacological levels COX-1 is expressed constitutively in all tissue Protective COX-2 is induced selectively in stimulated tissue Inflammatory Cyclooxygenase-2: A Proinflammatory Enzyme RA and atherosclerotic lesions Alzheimer’s disease Prostate carcinoma Colorectal carcinoma Angiogenesis Enhanced COX-2 Expression in Colon Cancer NORMAL NEOPLASTIC COX-2 (Prescott and Fitzpatrick, 2000, BBA) Se-deficiency Leads to Increased Expression of COX-2 COX-2 promoter NFkB mCOX-2/Luc -1000 -664 -396 TATA Box iNOS promoter NFkB miNOS/Luc -1742 -972 -86 TATA Box Selenium-Supplementation Decreases COX-2 Activity Macrophages stimulated with LPS (hours) -966 NF-kB NF-kB -668-59 -401-392 GGGAAATACC TCGATATGAC GGGGATTTCC GGTGTGTATC Se-Deficient Se-Supplemented COX-2 Luc COX-2-pGL3 Promoter Construct Zamamiri-Davis et al (2001) Free Radic. Biol. Med. COX-1 GAPDH + + - + + + - + LPS TLCK Nitric Oxide Synthase (Inducible) iNOS: • Generates NO by oxidation of L-arginine • Induced by a variety of immunologic and inflammatory mediators • Transcriptional activation of iNOS is a key mechanism for the regulation of NO production Dual Role in immune system: • Damaging vs. Defensive Production of Nitric Oxide by LPS-Activated Macrophage Hypotension Poor organ perfusion Hepatic dysfunction Islet cell death Nitrosylation of Proteins in Pathologies Associated with Human Diseases IHC: Anti-Nitrotyrosine Lung from a human patient with ARDS Atheromatous plaque in human artery Source: Crow and Beckman, 1995 Se-deficiency Increases the Expression and Activity of iNOS in LPS Stimulated RAW264.7 Macrophages RT-PCR IB Prabhu et al (2002) Biochem. J. iNOS Activity Activation of NF-kB leads to Increased Expression of iNOS iNOS PROMOTER ACTIVITY 4 Lucife rase Activity (fold induction) 3.5 3 SE- 2.5 SE+ 2 1.5 1 0.5 0 FL-iNOS WILD-TYPE M-iNOS DELETIONMUTANT Towards the Characterization of an Endogenous NF-kB Regulator Inactivation of the NF-kB Pathway by Se-Supplementation Se?? Se-Deficiency Leads to Increased levels of pIkBa IB:pSer IP:IkBa IB:pTyr IP:IkBa IB:IkBa IP:IkBa t= 0 (prior to LPS stimulation) IB Repletion of Se-deficient Cells with Se Lead to Decreased pIkBa levels Repleted SeLPS (h) 0 Se -/+ 2 0 MINUS 2 PLUS From Minus media to Plus media 1 Passage Activity of IKK is inhibited in Se-supplemented cells A Se-supplemented LPS(h) 0 0.5 1 2 Se-deficient 3 4 0 0.5 1 2 3 4 IB: Anti-p-Ser RAW 264.7 B LPS(h) BMDM IB: Anti-GST Se-supplemented 0 0.5 1 2 4 KA: p-IkBa-GST Se-deficient 0 0.5 1 2 4 IB: Anti-p-Ser IB: Anti-GST Negative Auto Regulatory Control of the NFkB Pathway IKKb specific inhibitors: A- and J-type PGs (IC50 = 2 mM) Rossi et al (2000) Nature Arachidonic Acid Metabolism Isoprostanes P450 Lipid peroxidation products LOX COX Mono & Di-hydroxy derivatives Mono & Di-hydroxy derivatives PGH2 PGF2a PGI2 PGE2 TXB2 HIV transcription Inflammation Allergic reactions PGD2 15d-PGJ2 VetSci 514/Nutr FitzGerald, G. A. et al. N Engl J Med 2001;345:433-442 PGD2 Metabolism PGDS PGH2 15d-PGJ2 • Se-supplementation of macrophages causes an increase in the production of 15d-PGJ2 15d-PGJ2 • Increased 15d-PGJ2 leads to inhibition of NF-kB-dependent pro-inflammatory gene expression • Inhibitory effect of 15d-PGJ2 mediated by inhibition of IKKb and activation of PPARg (transrepression) • 15d-PGJ2 formation in cells is Sedependent Vunta et al, (2007) J. Biol. Chem. 282: 17964-73 Requirement of SePr for the production of 15d-PGJ2 in macrophages Na2SeO3 (250 nM) Se- shSPS2 Se+ Vector Control IB: GPX-1 IB: TR1 AntiGAPDH Summary Se-deficiency leads to the production of several pro-inflammatory mediators (ROS) in cells. As a part of the antioxidant defense system, Se-supplementation decreases cellular oxidative stress. Deficiency of Se leads to increased expression of proinflammatory genes (COX-2, iNOS, TNFa) via the NF-kB pathway 15d-PGJ2 synthesis is dependent on cellular Se status and its synthesis is regulated by selenoproteins Inhibition of IKKb and activation of PPARg are both mediated via the direct modification by anti-inflammatory 15d-PGJ2 in Sesupplemented cells Summary Differential modulation of NF-kB and PPARg by Se could lead to the selective modulation of PG synthases Redox regulation can be viewed as another level of regulation superimposed on the more classical signal transduction events Thiol group modification in IKK by the endogenous a,b-unsaturated eicosanoid represents a previously undescribed mechanism of action of Se, which extends the code for redox regulation of proinflammatory gene expression