Regulation of Proinflammatory Gene Expression by Selenium

K. Sandeep Prabhu, Ph.D.Center for Molecular Toxicology & Carcinogenesis andCenter for Molecular Immunology & Infectious Disease

The Huck Institutes of the Life SciencesDepartment of Veterinary and Biomedical Sciences

The Pennsylvania State University, University Park, PAwww.vetsci.psu.edu

• Physiology and biochemistry of Selenium (Se)• Effect of Se on oxidative stress-induced pro-

inflammatory gene expression• Redox regulation of NF-B: Endogenous inhibitor

of NF-B in Se-supplemented mammalian cells• Regulation of the prostaglandin pathway

Chemistry of SeleniumDiscovered 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 0.12Skeleton 0.42 0.15Liver 0.54 0.78RBC 0.29 0.54Plasma 0.13 0.52Fatty 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

Prostate Cancer

Cardiovascular Diseases

Arthritis

Hypothyroidism

Colon Cancer

HIV

Selenium deficiency causes increased neutrophil adherence inbovine 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 outcomeof coliform mastitis

Mastitis

HIVCytokine levels of IL-2, TNF-, IL-8 decreased by Se-supplementation

Se impacts T-lymphocyte proliferation and differentiation

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.

Cardiomyopathy:

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

Se-Proteins SeCys SeMet

Selenate

SeleniteGSH

H2Se+Me

-MeCH3SeH

Selenoproteins

[H]

MeSeCys

TMSe Excretion

Metabolism of Inorganic and Organic Forms of Selenium

Selenide Methyl Selenol

L Y A S E S

Plants

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 α-helix immediately downstream of Sec (green box) in the selenoprotein sequences are indicated (right). Kryukov et al (2003) Science, 300(5624):1439-43

SelenoenzymesMammalian 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

proteinGPX6 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

[Se] M

Prevention of Keshan disease >0.25Optimal activity of IDs >0.82Maximization of plasma GPX, SePP >1.0-1.2Protection against some cancers >1.5

Source: Thomson CD (2004) Eu J Clin. Nutr. 58: 391-402

Assessment of Adequacy of Se Status:

NADP+

NADPH

2GSH

GSSG

Se-GPX

ROOH

ROH + H2O

NADP+

NADPH

Trx-(SH)2

Trx-S2

TrxR

ROOH

ROH + H2O

Selenoproteins•Selenium dependent Glutathione Peroxidase

•Thioredoxin reductase (TrxR)

Schematic of the VEGF-Mn-SOD signaling axis

FibroblastsT- & B-cellsEndothelial cells

NADPH OXIDASE (NOX)

PMA Receptor ligation

CytosolicSODs H2O2

ROS-Generating Enzymes

• NADPH Oxidase

• Cyclooxygenases (COX)

• Lipoxygenases (LOXs)

• SODs

• Fe & Cu-dependent enzymes (Fenton Chemistry)

Lipid intermediates

O2 O2.- H2O2

e-

O2 O2.- H2O2

Flavin-containingenzymes SOD

GPxCatalase

H2O + O2

.OH

Lipid-peroxidation

Damage toDNA/Protein

Mn-SOD

TrxR/Trx

e- e-e-e-

ONOO-

peroxynitrite

O2 O2.- H2O2

.OH + HO- H2O

oxygen superoxide hydrogen peroxide

hydroxyl radical

NO.

Reactive Oxygen Species (ROS)

ONOOHH+

peroxonitrous acid

Arginine

Citrulline

NOS

NADPH Oxidase

ROS/RNS

• 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

• DNA damage

• Lipid peroxidation

• Protein modification

Physiological functions

Cause of oxidative damage

AntioxidantsAntioxidants

ROS

REDOX STATUS

Increased ROS production results from an oxidant-antioxidant imbalance

SeleniumSeleniumVitamins C/EVitamins C/EGlutathione (GSH)Glutathione (GSH)Lipoic acidLipoic acidN-Acetyl Cys (NAC)N-Acetyl Cys (NAC)

HH2OO22, OH, OH--, O, O22.-.-, NO, NO..

Fatty acid hydroperoxidesFatty acid hydroperoxidesProstaglandinsProstaglandinsLeukotrienesLeukotrienes

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

Filled bars: 0.2 g Se/g diet as Na2SeO3; Open bars: 0.008 g Se/g diet; T= 28 days

Se-dependent GPX and TrxR activities are dependent onthe Se status in Rats

RAW264.7/Murine Bone Marrow-derived Macrophage Model

RAW264.7/BMDM in DMEM with 2 mM Gln,100 Units/ml Penicillin-G, 100 g/ml Streptomycin, 5 % FBS (defined) + M-CSF (50 ng/ml)

Se-deficient Se-supplemented(Se-) (Se+)

+ 0.25 - 2 nmol/ml Sodium SeleniteNo Se added

Depletion

Repletion

Mice (3 wk)

Se-deficient diet (0.01 ppm)

Se-supplemented diet (0.4 ppm)3 mo

GPX1 in Se-deficient and Se-supplemented cells

RAW264.7

BMDM

Se-Deficiency Increases Total ROS

Pathways ofNF-B Activation

Cell survival

Pro-inflammatory cytokines

Proinflammatory enzymes

Activation of PSA

Tumor initiation, promotion, andprogression

Inactivation of JNK

Adhesion molecules

NF-B Pathway

Copyright ©2005 American Society for Clinical InvestigationLuo, J.-L. et al. J. Clin. Invest. 2005;115:2625-2632

Inhibition of NF-B in cancer cells converts inflammation-induced tumor growth to tumor regression

CAPE: Inhibits the activation of NF-B

Natarajan et al (1996) PNAS (USA) 93, 9090-9095

•Isolated from propolis of honeybee hives•Potent and specific inhibitor of NF-kB activation induced by different agents•Mechanism is still unknown

Caffeic acid phenethyl ester

Guggulsterone inhibits NF-B activation

Shishodia S and Aggarwal BB (2004) J. Biol. Chem. 279, 47148-47158

Plant sterol from gum resinof Commiphora mukul

GS suppressed DNA bindingof NF-B induced by TNF,PMA, Okadaic acid, cig. Smoke

Mechanism: Inhibition of IKK activity?

Shishodia and Aggarwal, 2005

Activation of NF-B in Se-deficiency

Selenium-supplementation can inhibit the activation of NF-B in macrophages

kB LuckB kB kB kB

Luciferase reporter vectorZamamiri-Davis et al (2001) Free Radic. Biol. Med.

0 2 4 6 8 12 0 2 4 6 8 12IB:p65

IB: p50

Se-deficient Se-supplemented

Se-deficiency increases nuclear translocation of p65 and p50 proteins in HepG2 cells

LPS (h)

Se-Deficiency Exacerbates TNF 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

(Prescott and Fitzpatrick, 2000, BBA)

NORMAL NEOPLASTIC

COX-2

Se-deficiency Leads to Increased Expression of COX-2

COX-2 promoter

iNOS promoter

NFB

TATABox

mCOX-2/Luc-1000 -396-664

NFB

TATABox

miNOS/Luc

-86-972-1742

Macrophages stimulated with LPS (hours)

Selenium-Supplementation DecreasesCOX-2 Activity

-966

GAPDH

Se-Deficient Se-Supplemented

TLCK- + - +LPS

COX-2

COX-1

+ + + +

Zamamiri-Davis et al (2001) Free Radic. Biol. Med.

GGGAAATACCTCGATATGAC

GGGGATTTCCGGTGTGTATC

Luc-668-59 -401-392

NF-B NF-B

COX-2-pGL3Promoter Construct

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 keymechanism for the regulation of NO production

Dual Role in immune system:• Damaging vs. Defensive

Production of Nitric Oxide by LPS-Activated Macrophage

HypotensionPoor organ perfusionHepatic dysfunctionIslet cell death

Lung from a humanpatient with ARDS

IHC: Anti-Nitrotyrosine

Atheromatous plaquein human artery

Nitrosylation of Proteins in Pathologies Associated with Human Diseases

Source: Crow and Beckman, 1995

RT-PCR

IB

iNOS Activity

Se-deficiency Increases the Expression and Activity of iNOS in LPS Stimulated RAW264.7 Macrophages

Prabhu et al (2002) Biochem. J.

WILD-TYPE DELETION- MUTANT

Activation of NF-B leads to Increased Expression of iNOS

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 pIB

t= 0 (prior to LPS stimulation)

Se-defi

cient

Se-supplem

ented

IB:IBIP:IB

IB:pTyrIP:IB

IB:pSerIP:IB

IB

Repleted

MINUS PLUS

From Minus media to Plus media

Repletion of Se-deficient Cells with SeLead to Decreased pIB levels

Se- Se -/+0 2 0 2LPS (h)

1 Passage

KA: p-IB-GST

Activity of IKK is inhibited in Se-supplemented cells

IB: Anti-p-Ser

IB: Anti-GST

0 0.5 1 2 3 4 0 0.5 1 2 3 4

Se-supplemented Se-deficient

LPS(h)

0 0.5 1 2 4 0 0.5 1 2 4

Se-supplemented Se-deficient

RAW 264.7

BMDM IB: Anti-p-Ser

LPS(h)

A

B

IB: Anti-GST

Negative Auto Regulatory Control of the NFB PathwayIKK specific inhibitors: A- and J-type PGs (IC50 = 2 M)

Rossi et al (2000) Nature

Arachidonic Acid MetabolismArachidonic Acid Metabolism

COXLOX

PGH2

PGE2PGD2

PGF2

TXB2

PGI2

Mono & Di-hydroxyderivatives

P450

Mono & Di-hydroxyderivatives

HIV transcriptionInflammationAllergic reactions

Isoprostanes

Lipid peroxidationproducts

VetSci 514/Nutr15d-PGJ2

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

PGD2 Metabolism

PGH2

PGDS

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-B-dependent pro-inflammatory gene expression

• Inhibitory effect of 15d-PGJ2 mediated by inhibition of IKK and activation of PPAR (transrepression)

• 15d-PGJ2 formation in cells is Se-dependent

Vunta et al, (2007) J. Biol. Chem. 282: 17964-73

Requirement of SePr for the production of 15d-PGJ2 in macrophages

Se- shSPS2

Na2SeO3 (250 nM)

Vector Control

IB: GPX-1

Se+

IB: TR1

AntiGAPDH

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, TNF) via the NF-B pathway

15d-PGJ2 synthesis is dependent on cellular Se status and its synthesis is regulated by selenoproteins

Inhibition of IKK and activation of PPAR are both mediated via the direct modification by anti-inflammatory 15d-PGJ2 in Se-supplemented cells

Summary

Differential modulation of NF-B and PPAR by Se could lead to theselective 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 ,-unsaturated eicosanoid represents a previously undescribedmechanism of action of Se, which extends the code for redox regulation of proinflammatory gene expression

Summary