Regulation of Proinflammatory Gene Expression by Selenium K

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- B: Endogenous inhibitor of NF- B 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: 1 s 22 p 63 s 13 p 63 d 104 s 24 p 4 Valence Numbers: -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: 31 mcg 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) _____________________ 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 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 Se. Cys Se. Met LYASES Selenate Selenite Me. Se. Cys GSH [H] H 2 Se Selenide +Me -Me CH 3 Se. H 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): 14

Selenoenzymes Mammalian enzymes— Glutathione peroxidases (GPX) GPX 1 Classical glutathione peroxidase (GSH-Px) GPX 2 Gastrointestinal glutathione peroxidase (GPX-GI) GPX 3 Plasma glutathione peroxidase (Plasma GPX) GPX 4 Phospholipid hydroperoxide GPX (PHGPX) GPX 5 Androgen-regulated epididymal secretory protein GPX 6 Odorant-metabolizing protein Thioredoxin reductases (Trx. R 1 -4) Deiodonases (DI) DI 1 Iodothyronine 5’-deiodonase-1 (type 1 DI) DI 2 Iodothyronine 5’-deiodonase-1 (type 2 DI) DI 3 Iodothyronine 5’-deiodonase-1 (type 3 DI) Sel-P Plasma selenoprotein P

Assessment of Adequacy of Se Status: [Se] m. M Prevention of Keshan disease Optimal activity of IDs Maximization of plasma GPX, Se. PP 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 2 GSH NADP+ ROOH Se-GPX NADPH GSSG ROH + H 2 O • Thioredoxin reductase (Trx. R) Trx-(SH)2 NADP+ NADPH Trx. R Trx-S 2 ROOH ROH + H 2 O

PMA Receptor ligation NADPH OXIDASE (NOX) Fibroblasts T- & B-cells Endothelial cells Cytosolic SODs H 2 O 2 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 e- O 2 oxygen NO. NOS H 2 O 2 H+ ONOOperoxynitrite Citrulline e- OH + HO - e- H 2 O hydroxyl radical ONOOH Trx. R/Trx GPx H 2 O + O 2 O 2 . peroxonitrous acid Flavin-containing enzymes. - SOD O 2 e- hydrogen peroxide superoxide Arginine O 2 e- . - H 2 O 2 Catalase. Lipid-peroxidation OH . -Mn-SOD H 2 O 2 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 RE D STA OX TU S Antioxidants Selenium Vitamins C/E Glutathione (GSH) Lipoic acid N-Acetyl Cys (NAC) ROS H 2 O 2, OH-, O 2. -, NO. Fatty acid hydroperoxides Prostaglandins Leukotrienes q Cancer q AIDS q Arthritis q Atherosclerosis q Alzheimer's q Aging q Diabetes q 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 Trx. R activities are dependent on the Se status in Rats Filled bars: 0. 2 mg Se/g diet as Na 2 Se. O 3; Open bars: 0. 008 mg Se/g diet; T= 28 days

RAW 264. 7/Murine Bone Marrow-derived Macrophage Model Mice (3 wk) 3 mo Se-deficient diet (0. 01 ppm) Se-supplemented diet (0. 4 ppm) RAW 264. 7/BMDM in DMEM with 2 m. M 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+)

GPX 1 in Se-deficient and Se-supplemented cells RAW 264. 7 BMDM

Se-Deficiency Increases Total ROS

Pathways of NF- B Activation

NF-k. B Pathway Cell survival Pro-inflammatory cytokines Proinflammatory enzymes Adhesion molecules Activation of PSA Tumor initiation, promotion, and progression Inactivation of JNK

Inhibition of NF- B in cancer cells converts inflammation-induced tumor growth to tumor regression Copyright © 2005 American Society for Clinical Investigation Luo, J. -L. et al. J. Clin. Invest. 2005; 115: 2625 -2632

CAPE: Inhibits the activation of NF-k. B Caffeic acid phenethyl ester • Isolated from propolis of honeybee hives • Potent and specific inhibitor of NF-k. B activation induced by different agents • Mechanism is still unknown Natarajan et al (1996) PNAS (USA) 93, 9090 -9095

Guggulsterone inhibits NF-k. B activation Plant sterol from gum resin of Commiphora mukul GS suppressed DNA binding of NF-k. B induced by TNFa, PMA, Okadaic acid, cig. Smo Mechanism: Inhibition of IKK activity? Shishodia S and Aggarwal BB (2004) J. Biol. Chem. 279, 47148 -47158

Shishodia and Aggarwal, 2005

Activation of NF- B in Se-deficiency

Selenium-supplementation can inhibit the activation of NF- B in macrophages Luciferase reporter vector k. B k. B Zamamiri-Davis et al (2001) Free Radic. Biol. Med.

Se-deficiency increases nuclear translocation of p 65 and p 50 proteins in Hep. G 2 cells Se-deficient LPS 0 2 4 6 Se-supplemented 8 12 0 2 4 6 (h)8 12 IB: p 65 IB: p 50

Se-Deficiency Exacerbates TNFa Production in BMDM

COX Isoforms v Two known isoforms: COX-1 and COX-2 v Share 60% sequence homology, aspirin acetylation and glycosylation sites v 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 Ø RA and atherosclerotic lesions Ø Alzheimer’s disease Ø Prostate carcinoma Ø Colorectal carcinoma ØAngiogenesis

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 NF B -1000 m. COX-2/Luc -664 -396 TATA Box i. NOS promoter NF B mi. NOS/Luc -1742 -972 -86 TATA Box

Selenium-Supplementation Decreases COX-2 Activity Macrophages stimulated with LPS (hours)

-966 NF-k. B -668 -59 -401 -392 GGGAAATACC TCGATATGAC GGGGATTTCC GGTGTGTATC Se-Deficient Se-Supplemented COX-2 Luc COX-2 -p. GL 3 Promoter Construct Zamamiri-Davis et al (2001) Free Radic. Biol. Med. COX-1 GAPDH + + - + LPS TLCK

Nitric Oxide Synthase (Inducible) i. NOS: • Generates NO by oxidation of L-arginine • Induced by a variety of immunologic and inflammatory mediators • Transcriptional activation of i. NOS 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 üHypotension üPoor organ perfusion üHepatic dysfunction üIslet 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 i. NOS in LPS Stimulated RAW 264. 7 Macrophages RT-PCR IB Prabhu et al (2002) Biochem. J. i. NOS Activity

Activation of NF- B leads to Increased Expression of i. NOS WILD-TYPE DELETIONMUTANT

Towards the Characterization of an Endogenous NF- B Regulator

Inactivation of the NF- B Pathway by Se-Supplementation Se? ?

Se-Deficiency Leads to Increased levels of p. I Ba d e t n e m t n e i c fi de e S S le p p u s e IB: p. Ser IP: I Ba IB: p. Tyr IP: I Ba IB: I Ba IP: I Ba t= 0 (prior to LPS stimulation) I B

Repletion of Se-deficient Cells with Se Lead to Decreased p. I Ba levels Repleted Se. LPS (h) 0 MINUS Se -/+ 2 0 2 PLUS From Minus media to Plus media 1 Passage

Activity of IKK is inhibited in Se-supplemented cells A LPS(h) Se-supplemented 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-Ik. Ba-GST Se-deficient 0 0. 5 1 2 4 IB: Anti-p-Ser IB: Anti-GST

Negative Auto Regulatory Control of the NF B Pathway IKKb specific inhibitors: A- and J-type PGs (IC 50 = 2 m. M) Rossi et al (2000) Nature

Arachidonic Acid Metabolism Isoprostanes P 450 COX Mono & Di-hydroxy derivatives PGH 2 PGF 2 a PGI 2 PGE 2 Lipid peroxidation products LOX TXB 2 HIV transcription Inflammation Allergic reactions PGD 2 15 d-PGJ 2 Vet. Sci 514/Nutr

Fitz. Gerald, G. A. et al. N Engl J Med 2001; 345: 433 -442

PGD 2 Metabolism PGDS PGH 2

15 d-PGJ 2 • Se-supplementation of macrophages causes an increase in the production of 15 d-PGJ 2

15 d-PGJ 2 • Increased 15 d-PGJ 2 leads to inhibition of NF-k. B-dependent pro-inflammatory gene expression • Inhibitory effect of 15 d-PGJ 2 mediated by inhibition of IKKb and activation of PPARg (transrepression) • 15 d-PGJ 2 formation in cells is Sedependent Vunta et al, (2007) J. Biol. Chem. 282: 17964 -73

Requirement of Se. Pr for the production of 15 d-PGJ 2 in macrophages Na 2 Se. O 3 (250 n. M) Se- sh. SPS 2 Se+ Vector Control IB: GPX-1 IB: TR 1 Anti. GAPDH

Summary ØSe-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. ØDeficiency of Se leads to increased expression of proinflammatory genes (COX-2, i. NOS, TNFa) via the NF-k. B pathway Ø 15 d-PGJ 2 synthesis is dependent on cellular Se status and its synthesis is regulated by selenoproteins ØInhibition of IKKb and activation of PPARg are both mediated via the direct modification by anti-inflammatory 15 d-PGJ 2 in Sesupplemented cells

Summary ØDifferential modulation of NF-k. B and PPARg by Se could lead to the selective modulation of PG synthases ØRedox regulation can be viewed as another level of regulation superimposed on the more classical signal transduction events ØThiol 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