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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