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