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Free Radical Biology and Medicine 53 (2012) 629–640
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Free Radical Biology and Medicine
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Abbre
molecu
factor-kreactive
nuclear
associat
NAD(P)
subunit
S-transf
tin-protn Corr
Laborat
Chunch
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journal homepage: www.elsevier.com/locate/freeradbiomed
Original Contribution
Functional dissection of Nrf2-dependent phase II genes in vascularinflammation and endotoxic injury using Keap1 siRNA
Ji-Hee Kim a, Yoon Kyung Choi a, Kwang-Soon Lee a, Dong-Hui Cho a, Yi-Yong Baek a,Dong-Keon Lee a, Kwon-Soo Ha a, Jongseon Choe b, Moo-Ho Won c, Dooil Jeoung d, Hansoo Lee e,Young-Guen Kwon f, Young-Myeong Kim a,n
a Vascular Homeostasis Laboratory and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Kangwon-do 200-701,
Koreab Department of Microbiology, School of Medicine, Kangwon National University, Chunchon, Kangwon-do 200-701, Koreac Department of Neurobiology, School of Medicine, Kangwon National University, Chunchon, Kangwon-do 200-701, Koread Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chunchon, Kangwon-do 200-701, Koreae Department of Biological Sciences, College of Natural Sciences, Kangwon National University, Chunchon, Kangwon-do 200-701, Koreaf Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea
a r t i c l e i n f o
Article history:
Received 20 January 2012
Received in revised form
8 April 2012
Accepted 20 April 2012Available online 27 April 2012
Keywords:
Keap1
Nrf2
Phase II gene
NF-kB
Inflammation
Free radicals
49/$ - see front matter & 2012 Elsevier Inc. A
x.doi.org/10.1016/j.freeradbiomed.2012.04.01
viations: TNF-a, tumor necrosis factor-a; ICA
le-1; VCAM-1, vascular cell adhesion molecul
B; IkB, inhibitory kB; GSH, glutathione; GSSG
oxygen species; IKK, IkB kinase; NIK, NF-kB
factor-erythroid 2p45 (NF-E2)-related factor
ed protein 1; ARE/EpRE, antioxidant/electroph
H:quinone oxidoreductase 1; GCLC, glutamat
; GCLM, glutamate–cysteine ligase modifier s
erase Mu 1; HO-1, heme oxygenase-1; Prx1,
oporphyrin IX; BSO, l-buthionine-S-sulfoximi
esponding author at: Kangwon National Univ
ory, Department of Molecular and Cellular Bioc
on, Kangwon-do 200-701, Korea. Fax: þ82 33
ail address: [email protected] (Y.-M. Kim
a b s t r a c t
Keap1 is a cytoplasmic repressor of the transcription factor Nrf2, and its degradation induces Nrf2
activation, leading to upregulation of antioxidant phase II genes. We investigated the roles of phase II
genes in vascular inflammation and septic injury using Keap1 siRNA and elucidated its underlying
mechanism. Selective knockdown of Keap1 with siRNA promoted Nrf2-dependent expression of phase
II genes in endothelial cells, such as heme oxygenase-1 (HO-1), glutamate–cysteine ligase (GCL), and
peroxiredoxin-1 (Prx1), resulting in the elevation of cellular glutathione levels and suppression of
tumor necrosis factor (TNF)-a-induced intracellular H2O2 accumulation. Keap1 knockdown inhibited
TNF-a-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion
molecule-1 (VCAM-1) by suppressing NF-kB activation via inhibition of its upstream modulators, Akt,
NIK, and IKK, resulting in the elevation of monocyte adhesion to endothelial cells. Importantly, these
events were reversed by HO-1 and GCL inhibitors and Prx1-specific siRNA. Keap1 knockdown also
inhibited endotoxin-induced expression of inducible nitric oxide synthase (iNOS) and TNF-a by
upregulating HO-1, GCL, and Prx1 expression in macrophages. Moreover, in vivo Keap1 knockdown
increased the expression of phase II genes and suppressed the expression of ICAM-1, VCAM-1, iNOS,
and TNF-a in an endotoxemic mouse model, resulting in significant protection against liver and lung
injuries and lethality. Our results indicate that Keap1 knockdown prevents NF-kB-mediated vascular
inflammation and endotoxic shock by suppressing NF-kB-mediated inflammatory gene expression via
upregulation of Nrf2-mediated antioxidant genes. Thus, siRNA targeting Keap1 may provide a new
therapeutic approach for inflammation-associated vascular diseases and sepsis.
& 2012 Elsevier Inc. All rights reserved.
ll rights reserved.
9
M-1, intercellular adhesion
e-1; NF-kB, nuclear
, oxidized glutathione; ROS,
inducing kinase; Nrf2,
2; Keap1, Kelch-like-ECH-
ile response element; NQO1,
e–cysteine ligase catalytic
ubunit; GSTM1, glutathione
peroxiredoxin-1; SnPP,
ne; LPS, lipopolysaccharide
ersity, Vascular Homeostasis
hemistry, School of Medicine,
244 3286.
).
Introduction
Inflammation is closely associated with the pathogenesis ofmany human diseases, including atherosclerosis, cardiovasculardiseases, and sepsis. Endothelial cells are important participantsin vascular inflammation [1]. Inflammatory stimuli, includingtumor necrosis factor-a (TNF-a), increase the expression ofadhesion molecules, such as intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), on thesurface of endothelial cells, which bind circulating immune cellsresulting in leukocyte recruitment and infiltration, cytokineproduction, and induction of tissue inflammation [2–4]. Theexpression of adhesion molecules and other inflammation-asso-ciated genes, such as TNF-a and inducible nitric oxide synthase
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640630
(iNOS), is tightly regulated by the activation of nuclear factor-kB(NF-kB). Therefore, selective inhibition of NF-kB activation/activ-ity prevents hyperinflammation and is used as a therapeutictreatment for various human inflammatory diseases [5,6].
Keap1 (Kelch-like ECH-associated protein 1) is a cytoplasmicrepressor protein essential for complex formation with the tran-scription factor Nrf2, leading to inhibition of its nuclear trans-location [7]. Keap1 contains redox-sensitive cysteine residues atpositions 273 and 288 and is associated with ubiquitin E3 ligase(Cul3), which is responsible for ubiquitination of Nrf2. Under normalconditions, Keap1 promotes Nrf2 degradation via the Cul3-depen-dent ubiquitin–proteasome pathway [7]. However, upon exposureto oxidative or electrophilic stress, the reactive cysteine residues ofKeap1 become modified, leading to a decline in E3 ligase activity,stabilization of Nrf2, and its subsequent translocation into nucleus.This translocation leads to heterodimer formation with small MAFproteins that bind to the antioxidant/electrophile response element(ARE/EpRE) sequence in the promoters of phase II genes, whichstimulates their expression.
Among the phase II enzymes, heme oxygenase-1 (HO-1),glutamate–cysteine ligase catalytic subunit (GCLC), glutamate–cysteine ligase modifier subunit (GCLM), peroxiredoxin-1 (Prx1),NAD(P)H:quinone oxidoreductase 1 (NQO1), and glutathione S-transferase Mu 1 (GSTM1) play important roles in maintainingredox homeostasis and regulating inflammatory response [8].Indeed, some phase II gene products, including HO-1, GCL, andPrx1, have been demonstrated to suppress intracellular accumu-lation of reactive oxygen species (ROS), which play an importantrole in the redox-sensitive NF-kB activation pathway [9–12].Thus, these enzymes/proteins are critically involved in the reg-ulation of NF-kB-dependent inflammatory gene expression. Theseobservations indicate that phase II enzymes contribute to theregulation of NF-kB-mediated inflammatory responses. Manycytoprotective and anti-inflammatory compounds have beenshown to suppress cytotoxic gene expression mostly by upregu-lation of HO-1 expression; however, the protective role of addi-tional Nrf2-dependent genes has not been extensively studied.We hypothesize that Keap1 downregulation promotes Nrf2-induced gene expression and inhibits vascular inflammation andleukocyte infiltration into inflamed sites by suppressing theexpression of inflammatory cytokine and adhesion moleculeexpression, resulting in the prevention of human inflammatorydiseases.
In this study, we investigated the effects of short interferingRNA (siRNA)-mediated Keap1 knockdown on inflammation andcytoprotection in both cell culture and an animal model toelucidate its underlying mechanism in these processes. Ourresults showed that Keap1 knockdown inhibited NF-kB activationand inflammatory gene expression in macrophages and endothe-lial cells by increasing Nrf2-dependent expression of ARE/EpRE-regulatory phase II genes, particularly HO-1, GCL, and Prx1,resulting in protection of mice from LPS-induced organ injuryand lethality.
Materials and methods
Chemicals and reagents
Medium 199 (M199), fetal bovine serum (FBS), penicillin, andstreptomycin were purchased from Thermo Fisher Scientific(Waltham, MA, USA). RPMI 1640, Dulbecco’s modified Eaglemedium (DMEM), and TRIzol reagent kit were purchased fromInvitrogen (Grand Island, NY, USA). Recombinant human TNF-aand ELISA kit for TNF-a were obtained from R&D Systems(Minneapolis, MN, USA). Basic fibroblast growth factor was
obtained from Upstate Biotechnology (Lake Placid, NY, USA).Luciferase assay system, poly(dI–dC), and NF-kB-specific oligo-nucleotide were purchased from Promega (Madison, WI, USA).Antibodies for iNOS, p-IKKa/b, HO-1, and PARP were purchasedfrom BD Transduction Laboratories (San Diego, CA, USA), CellSignaling Technology (Beverly, MA, USA), Stressgen (Madison, WI,USA), and Calbiochem (San Diego, CA, USA), respectively. Otherantibodies used were obtained from Santa Cruz Biotechnology(Santa Cruz, CA, USA). 20, 70-Dichlorofluorescin diacetate (DCFH2-DA) was purchased from Molecular Probes (Eugene, OR, USA). ThesiRNAs for Keap1, Prx1, and GSTM1 were obtained from SantaCruz Biotechnology. Other chemicals were purchased from Sigma(St. Louis, MO, USA).
Cell culture
Human umbilical vein endothelial cells (HUVECs) isolatedfrom human umbilical cord veins were cultured as previouslydescribed [13] and used for experiments in passages 3 to 6. Theimmortalized murine macrophage RAW264.7 cells were grown inDMEM supplemented with 5% FBS, 100 U/ml penicillin, 100 mg/mlstreptomycin, and 25 mM Hepes at 37 1C in humidified 5% CO2/95% air. U937 cells were grown in RPMI 1640 containing 10% FBS,100 U/ml penicillin, and 100 mg/ml streptomycin in a CO2 incu-bator. HUVECs and RAW264.7 cells were transfected respectivelywith 100 nM human and mouse siRNA for Keap1, Prx1, or GSTM1using a microporator (Digital Bio Technology, Suwon, Korea). Cellswere pretreated with 20 mM enzyme inhibitors (tin-protopor-phyrin IX (SnPP), buthionine sulfoximine (BSO), and dicumarol)for 1 h before stimulation with TNF-a (10 ng/ml) or LPS (1 mg/ml).
Measurement of NO, TNF-a, ROS, and GSH
The levels of nitrite and total nitrite plus nitrate (NOx), stableoxidized products of NO, were measured in culture media andsera using Griess reagents and a reductase-based colorimetricassay kit (Alexis, San Diego, CA, USA), respectively. The serumlevel of TNF-a was determined in culture media and sera using anELISA kit (R&D Systems). Intracellular ROS accumulation wasdetermined using the H2O2-sensitive fluorescent dye DCFH2-DA[14]. HUVECs transfected with Keap1 siRNA for 24 h werepretreated with 20 mM SnPP, BSO, or dicumarol for 1 h, followedby stimulation with 10 ng/ml TNF-a for 1 h. Cells were furtherincubated with DCFH2-DA (10 mM) for 30 min. Cells were washedtwice with phosphate-buffered saline (PBS), and intracellularlevels of ROS were analyzed by confocal microscopy. AfterHUVECs were treated with TNF-a for 1 h, levels of GSH and totalGSH (GSHþGSSG) were determined in cell lysates using aglutathione detection kit from Abcam (Cambridge, MA, USA).
Western blot analysis
Whole-cell lysates as well as cytosolic and nuclear fractionswere prepared as previously described [14]. Liver and lung tissueswere homogenized in ice-cold protein-extraction buffer containing100 mM Hepes (pH 7.9), 10% glycerol, 5% Triton X-100, 250 mMNaF, 5 mM Na3VO4, and a protease inhibitor cocktail (10 mg apro-tinin, 10 mg leupeptin, and 1 mM phenylmethanesulfonyl fluoride).The homogenates were centrifuged at 15,000g at 4 1C for10 min, and supernatants were collected for detecting specificproteins. Samples (50 mg protein) were electrophoretically sepa-rated on SDS–PAGE and transferred onto nitrocellulose mem-branes. Western blot was performed as previously described [14].
Table 1Sequences of RT-PCR oligonucleotide primers.
Species Gene Sense Antisense
Human Keap1 CATCCACCCTAAGGTCATGGA GACAGGTTGAAGAACTCCTCC
Human HO-1 CAGGCAGAGAATGCTGAG GCTTCACATAGCGCTGCA
Human GCLC TACGGAGGAACAATGTCCGA CAGTGTGAACCCAGGACAGC
Human GCLM GCTGTATCAGTGGGCACAGG TGACCGAATACCGCAGTAGC
Human GSTM1 GCCAGTGGCTGAATGAAAAA CCAGCTGCATATGGTTGTCC
Human GSTA4 GGAGTCCGTGAGATGGGTTT TGTTGGAACAGCAGGTGGTT
Human Prx1 GAGGACTGGGACCCATGAAC CAGTGAACTGGAAGGCCTGA
Human GAPDH CGCCACAGTTTCCCGGAGGG CCCTCCAAAATCAAGTGGGG
Mouse Keap1 GGAATGAGTGGCGGATGATCAC GCTTCAGCAGGTACAGTTTTG
Mouse HO-1 GGCCCTGGAAGAGGAGATA GCTGGATGTGCTTTTGGTG
Mouse GCLC CACTGCCAGAACACAGACCC ATGGTCTGGCTGAGAAGCCT
Mouse GCLM GGGCACAGGTAAAACCCAAT GCTTCCTGGAAACTTGCCTC
Mouse Prx1 CTTTTGTGTGTCCCACGGAG TGGGTGTGTTAATCCATGCC
Mouse ICAM-1 GTGGGTCGAAGGTGGTTCTT GCAGTTCCAGGGTCTGGTTT
Mouse VCAM-1 GTTGGGGATTCGGTTGTTCT AGAGCTCAACACAAGCGTGG
Mouse TNF-a ATGAGCACAGAAAGCATG TCACAGAGCAATGACTCC
Mouse iNOS TTTGGAGCAGAAGTGCAAAGTCTC GATCAGGAGGGATTTCAAAGACCT
Mouse Actin TCCTTCGTTGCCGGTCCACA CGTCTCCGGAGTCCATCACA
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640 631
Reverse transcription–polymerase chain reaction (RT-PCR)
Isolated RNA and complementary DNA were prepared asdescribed previously [14]. PCR was performed in 50 mM KCl,10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 0.2 mM dNTPs, 2.5 unitsof Taq DNA polymerase, and 0.1 mM each target primer for humanand mouse (Table 1). Amplification conditions were as follows:denaturation at 94 1C for 5 min for the first cycle and for 45 sstarting from the second cycle; annealing of human HO-1 at 60 1Cfor 30 s; annealing of human Keap1 and GAPDH and mouse HO-1,Keap1, and actin at 56 1C for 30 s; annealing of NQO1, GCLC,GCLM, GSTM1, GSTA4, ICAM-1, and VCAM-1 at 55 1C for 30 s;annealing of mouse iNOS at 65 1C for 1 min; and annealing ofmouse TNF-a at 51 1C for 45 s; and extension at 72 1C for 45 s for30 cycles. Final extension was performed at 72 1C for 10 min, andthe PCR products were determined by agarose gel electrophoresis.
Analyses of Nrf2 and NF-kB activation pathways
Activation pathways of the transcription factors Nrf2 and NF-kB were analyzed according to our previous report [14]. Electro-phoretic gel mobility-shift assay (EMSA) was performed forthe measurement of binding activities of Nrf2 and NF-kB to32P labeled consensus oligonucleotides in the nuclear extractsfrom HUVECs, which were cotransfected with 100 nM Keap1siRNA for 24 h or further treated with 10 ng/ml TNF-a in thepresence of 20 mM enzyme inhibitors. Specific binding of tran-scription factors was analyzed in the reaction mixtures, whichwere preincubated with an excess (�100) of cold probe or anti-body for each transcription factor at room temperature for30 min. Luciferase reporter gene assay was measured in lysatesfrom cells cotransfected with 1 mg/ml pGL3-ARE/EpRE-Luc (orpGL3-HO-1-Luc) and 100 nM Keap1 siRNA for 36 h, as well as inlysates from cells treated with TNF-a for 12 h in the presence orabsence of 20 mM enzyme inhibitors after cotransfection with1 mg/ml pGL3-NF-kB-Luc and 100 nM Keap1 siRNA for 24 h.Nuclear translocation of Nrf2 and NF-kB was examined using animmunocytochemical method. Briefly, after transfection withKeap1 siRNA for 24 h, cells were pretreated with 20 mM enzymeinhibitors, followed by simulation with TNF-a for 1 h. Cells werefixed with 3.7% formaldehyde and permeabilized with 0.1%saponin. Cells were incubated with rabbit polyclonal antibodies(1:100) for Nrf2 and NF-kB for 2 h at 4 1C and then incubated withanti-rabbit IgG–TRITC (1:200; Sigma). For nuclear staining, cellswere further incubated for 30 min with DAPI (1 mg/ml; Sigma).
After mounting, nuclear translocation of transcription factors wasobserved by confocal microscopy.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed as previously described [14].To analyze the binding activity of Nrf2 to the HO-1 promoter,HUVECs were transfected with Keap1 siRNA for 24 h. For assayingbinding activity of NF-kB to promoters of ICAM-1 and VCAM-1,HUVECs were transfected with Keap1 siRNA for 24 h and pre-treated with 20 mM enzyme inhibitors for 1 h, followed bystimulation with TNF-a for 1 h. Both cell lysates were preparedfor ChIP assay and immunoprecipitated with an antibody againstNrf2 or NF-kB p65. Immunoprecipitated DNA samples werepurified by phenol extraction. Targeted promoter sequences ofHO-1, ICAM-1, and VCAM-1 were identified by PCR (30 cycles at94 1C for 30 s, 55 1C for 30 s, 72 1C for 30 s) using primer pairsspanning ICAM-1- and VCAM-1-specific promoter regions con-taining the NF-kB-binding sequence and HO-1-specific promoterregions containing the ARE/EpRE-binding sequence. Productswere identified on a 2% agarose gel. The primer sequences wereas follows: forward 50-AGAAATGCCCGTGTCAGCTA-30 and reverse50-CCGAACTCAAGTCCTCCCTC-30 for ICAM-1; forward 50-GCCAG-GACAGAGAGAGGAGC-30 and reverse 50-CCTTCAAGGGGAAACC-CAG-30 for VCAM-1; forward 50-GGGATTAAACCTGGAGCAGC-30
and reverse 50- TTTTTCCTGCTGAGTCACGG-30 for HO-1.
HO activity assay
HO activity in endothelial cells was measured by bilirubingeneration [15]. Cells were transfected with Keap1 siRNA using amicroporator and recovered in fresh medium for 24 h, followedby further incubation with 20 mM SnPP. The cells were thenwashed with PBS and harvested. Cell pellets were suspended inMgCl2 (2 mM)–phosphate (100 mM) buffer (pH 7.4), lysed bythree cycles of freezing and thawing, and centrifuged at 12,000 g
for 15 min at 4 1C. The supernatant was added to a reactionmixture containing NADPH (0.8 mM), mouse liver cytosol (2 mg)as a source of biliverdin reductase, the substrate hemin (10 mM),glucose 6-phosphate (2 mM), and glucose-6-phosphate dehydro-genase (0.2 units) in a final volume of 400 ml. The reaction wasperformed in the dark for 1 h at 37 1C and the bilirubin formedwas extracted with chloroform (400 ml) and calculated by thedifference in absorbance between 464 and 530 nm using the
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640632
extinction coefficient of 40 mM�1 cm�1 for bilirubin. HO activitywas expressed as pmol of bilirubin formed/mg of protein/h.
Monocyte adhesion assay
HUVECs transfected with or without Keap1 siRNA were platedon 0.2% gelatin-coated 24-well plates at a density of 1�104 cells/well. After 36 h, cells were treated with TNF-a for 8 h aftertreatment with chemical inhibitors for 1 h. Cells were washedthree times with PBS, replenished with M199 containing 5% FBS,and incubated with human U937 cells (5�104 cells/well) for30 min. After three washes with PBS, the attached cells werefixed, stained with Diff-Quick solution (Baxter Healthcare, IL,USA), and then counted in five randomly selected microscopicfields for each well [16].
Flow cytometric analysis
HUVECs transfected with or without Keap1 siRNA for 24 hwere pretreated with 20 mM enzyme inhibitors for 1 h, followedby simulation with TNF-a for 12 h. After being washed twice withPBS, HUVECs were detached gently by treating with PBS contain-ing 10 mM EDTA. After as wash with PBS, the cells were blockedwith 3% bovine serum albumin for 1 h and then washed two timeswith PBS. Subsequently, the cells were incubated with rabbitpolyclonal antibody (1:50) for ICAM-1 or VCAM-1 for 1 h at 41C.The cells were washed and incubated with anti-rabbit IgG–TRITC(1:100) for 30 min in ice-cold PBS containing 2% bovine serumalbumin, fixed with 2% paraformaldehyde, and analyzed by flowcytometry in a fluorescence-activated cell sorter.
AsiRNA (nM)
0 50 100
Ke
ap
1N
rf2
Cyto
so
lic
fracti
on
Nu
cle
ar
fracti
on
siRNA (nM)
0 50 100
B
siRNA (nM)
p32-proble
Cold probe
Nrf2 ►
0
+
-
50
+
-
100
+
-
100
+
+
HO-1
input
0
siR
ED
0
-
-
Fig. 1. Transfection of HUVECs with Keap1 siRNA increases ARE/EpRE promoter activit
Keap1 siRNA using a microporator and allowed to recover in fresh medium for 30 h. (A)
separated on SDS–PAGE. Target proteins were determined by Western blot analysis. (C)
Nrf2 was determined by immunohistochemistry using an antibody for Nrf2 and DAPI.
presence or absence of cold probe. (E) Soluble chromatin from cells was immunoprecip
HO-1 promoter was amplified by PCR. (F) Luciferase gene reporter activity was analyze
EpRE-Luc for 48 h. The data shown are the means7SD (n¼3). **Po0.01 versus contro
Animal treatment
Male BALB/c mice (6 to 8 weeks of age) were obtained fromOrient (Sungnam, Korea) and maintained at the specific-patho-gen-free housing facility. All procedures performed on theseanimals were in accordance with the guidelines of the UniversityAnimal Care and Use Committee. For lethality assay, mice wereinjected i.p. with LPS (35 mg/kg) at 24 h after intravenous injec-tion with a mixture (400 ml) of Keap1 siRNA (0.5 mg/kg) andin vivo-jetPEI reagent (Polyplus-Transfection, NY, USA). Lethalitywas monitored every 6 h. In addition, whole blood and organswere obtained from mice treated with LPS for 12 h by cardiacpuncture and surgical operation. The organs and serum preparedfrom whole blood by centrifugation at 3000 g at 41C were quicklyfrozen and kept at �701C until use.
Histological examination
Mouse liver and lung tissues were fixed in 10% formaldehydeand embedded in paraffin. Five-micrometer-thick tissue sectionswere placed on polylysine-coated slides and stained with hema-toxylin and eosin. The slides were examined under an opticalmicroscope.
Statistical analysis
The data are presented as means7standard deviation (SD) ofat least three separate experiments. Comparisons between twogroups were analyzed using the Student t-test, and significancewas established at Po0.05.
CNrf2 DAPI Merge
05
01
00
siR
NA
(n
M)
4
RL
A (
fold
)
3
2
1
0
siRNA (nM)
0 50 100
**
**
F
Nrf
2A
cti
nP
AR
PN
rf2
50 100
NA (nM)
y by stabilizing Nrf2 and its nuclear translocation. HUVECs were transfected with
Whole-cell lysates and (B) cytosolic and nuclear fractions were electrophoretically
Transfected cells recovered in fresh medium for 24 h; nuclear translocalization of
(D) Nuclear extracts were analyzed for Nrf2–DNA binding activity by EMSA in the
itated with anti-Nrf2 antibody. The region of the ARE/EpRE sequence of the human
d in lysates from cells cotransfected with 100 nM Keap1 siRNA and 1 mg/ml pARE/
l.
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640 633
Results
Specific knockdown of Keap1 increases Nrf2 activation
We first conducted experiments to determine the effects of theKeap1 siRNA on Nrf2 stabilization and its translocation into thenucleus, where it activates the transcriptional expression of phaseII enzymes. Transfection of HUVECs with Keap1 siRNA inhibitedKeap1 protein expression and elevated the level of Nrf2 protein in adose-dependent manner (Fig. 1A). Western blot and immunohis-tochemical analyses revealed that Keap1 knockdown decreased thecytosolic level of Nrf2 compared with control, resulting in anincrease in its nuclear level (Fig. 1B and C). EMSA revealed asignificant increase in DNA-binding activity of Nrf2 in nuclearextracts from HUVECs transfected with Keap1 siRNA, and thisbinding specificity was confirmed by competition assay using anexcessive amount of cold probe (Fig. 1D). Moreover, ChIP assay alsoshowed an increase in functional binding of Nrf2 to the HO-1promoter in Keap1 siRNA-transfected HUVECs (Fig. 1E). We nextexamined the effect of Keap1 knockdown on the transcriptionalactivity of Nrf2 using an ARE/EpRE-luciferase reporter gene.Transfection with Keap1 siRNA resulted in a significant increasein the transcriptional activity of the ARE/EpRE promoter in a dose-dependent manner compared with control cells (Fig. 1F). Thesedata suggest that Keap1 knockdown increases the expression ofphase II genes by promoting Nrf2 activation and its translocationinto the nucleus.
Keap1 knockdown increases the expression of ARE/EpRE-regulatory
phase II genes in HUVECs
We determined the effect of Keap1 knockdown on the mRNAlevels of Nrf2-dependent phase II genes, such as NQO1, GCLC,GCLM, GSTM1, GSTA4, and HO-1. Keap1 knockdown increased themRNA levels of these specific genes (Fig. 2A) and also upregulatedthe protein level of HO-1, a typical target of phase II genes (Fig. 2B).
B
siRNA
HO-1
Actin
-
C
40
20
0
To
tal G
SH
(n
mo
le/m
g p
rote
in)
siRNA
BSO
-
-
60 *E
siR
A
siRNA -
NQO1
GCLC
GCLM
GSTM1
GSTA4
GAPDH
HO-1
Keap1
Fig. 2. Keap1 knockdown increases ARE/EpRE-regulatory phase II gene expression in HU
recover in fresh medium for 30 h. (A) The mRNA levels of ARE/EpRE-regulated phase II g
Western blot analysis. (C) Cells were cotransfected with 100 nM Keap1 siRNA and 1 mg/
a luminometer. (D–F) Cell lysates were prepared from HUVECs, which were transfected
(D) HO-1 activity in the lysates was measured in the presence or absence of 20 mM SnP
using a GSH assay kit. The data shown in graphs are the means7SD (n¼3). **Po0.01
Furthermore, cells transfected with Keap1 siRNA showed a signifi-cant increase in HO-1 promoter and enzyme activities, and itscatalytic activity was inhibited by treatment with the HO-1inhibitor SnPP (Fig. 2C and D). Because Keap1 knockdown increasedthe transcriptional expression of GCL, a rate-limiting enzyme inGSH synthesis, we examined intracellular GSH levels in HUVECstransfected with Keap1 siRNA, by using a GSH assay kit. Keap1knockdown increased intracellular levels of total GSH (GSHþGSSG)and reduced GSH, and these effects were reversed by the GCLinhibitor BSO (Fig. 2E and F). These results indicate that Keap1knockdown induces antioxidant gene expression.
Keap1 knockdown suppresses TNF-a-induced adhesion molecule
expression
We next examined whether Keap1 knockdown regulates theexpression of vascular adhesion molecules, such as ICAM-1 andVCAM-1, which play an important role in vascular inflammatoryresponses as well as the pathogenesis of a variety of inflammatorydiseases [3]. Keap1 knockdown suppressed TNF-a-mediatedincreases in the total protein and mRNA levels of ICAM-1 andVCAM-1 in HUVECs, and these suppressive effects were reducedby treatment with HO-1 inhibitor (SnPP) and GCL inhibitor (BSO),but not with NQO1 inhibitor (dicumarol) (Fig. 3A). We did notfind any cytotoxic effects of these inhibitors under our experi-mental conditions (data not shown). We further examined theeffect of Keap1 siRNA on the cell surface levels of these adhesionmolecules in TNF-a-stimulated HUVECs using flow cytometricanalysis. Keap1 knockdown suppressed TNF-a-induced increasesin cell surface levels of ICAM-1 and VCAM-1 in HUVECs, and theseincreases were reduced by treatment with SnPP and BSO, but notwith dicumarol (Fig. 3B–D). It is well known that the adhesion ofleukocytes from circulating blood to vascular endothelial cells isthe earliest and most essential process in vascular inflammatoryresponses as well as in the initiation of vascular diseases [3].As such, we examined whether Keap1 knockdown would regulate
D
40
30
20
0
GS
H
(nm
ole
s/m
g p
rote
in)
siRNA
BSO
-
-
+
+
+
-
50
+
-
+
+
* ** ** **F
siRNA
SnPP
-
-
+
-
+
+
0
HO
-1 a
cti
vit
y(n
mo
l o
f B
RB
/mg
/h) 15
10
5
** **
Lu
c a
cti
vit
y (
fold
) 6
4
2
0NA -
**
**
10
VECs. HUVECs were transfected with 50 and 100 nM Keap1 siRNA and allowed to
enes were determined by RT-PCR. (B) The level of HO-1 protein was determined by
ml pGL3-HO-1-Luc for 48 h. Luciferase activity was measured in cell extracts using
with Keap1 siRNA for 24 h and incubated with 20 mM SnPP or 20 mM BSO for 12 h.
P. The levels of (E) total GSH and (F) reduced GSH were determined in cell lysates
versus control.
TNF-αsiR-Keap1
Inhibitor - - -
- + + + + +
- - + + + +
ICAM-1
VCAM-1
Actin
ICAM-1
VCAM-1
GAPDH
+
-
-
+
+
-
300
200
100
0
MC
F (
%)
TNF-αsiR-Keap1
Inhibitor
-
-
-
+
+
+
+
+
+
-
-
-
TNF-αsiR-Keap1
Inhibitor
+
-
-
+
+
-
+
+
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+
300
200
100
0
MC
F (
%)
+
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TNF-αsiR-Keap1
Inhibitor
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0Mo
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cyte
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) 12
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ba
c
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100 101 10210
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60
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100
FL2-H
Co
un
ts
CTRL
TNFTNF+siRNA
TNF+siRNA+BSO
TNF+siRNA+SnPP
****
*
****
******
**
SnPP
BSO
DC
SnPP
BSODC
SnPP
BSODC
SnPP
BSODC
Fig. 3. Keap1 knockdown suppresses TNF-a-induced adhesion molecule expression. HUVECs were transfected with 100 nM Keap1 siRNA and allowed to recover in fresh
medium for 30 h. (A) Transfected cells were treated with TNF-a (10 ng/ml) for 12 h after pretreatment with 20 mM SnPP, 20 mM BSO, and 20 mM dicumarol (DC) for 1 h. The
levels of ICAM-1 and VCAM-1 protein and mRNA were determined by Western blotting and RT-PCR. Under the same experimental conditions, the cell surface levels of
(B and C) ICAM-1 and (D) VCAM-1 were determined by flow cytometric analysis. Data shown are the relative mean channel fluorescence (MCF)7SD (n¼3). *Po0.05 and
**Po0.01. (E and F) Transfected cells were incubated with TNF-a for 12 h in the presence or absence of SnPP, BSO, and DC. Cells were incubated with U937 cells for 30 min,
and the levels of monocyte adhesion were determined after staining with Diff-Quick solution. (a) Control, (b) TNF-a, (c) siRNAþTNF-a, (d) siRNAþTNF-aþSnPP,
(e) siRNAþTNF-aþBSO, (f) siRNAþTNF-aþDC. Data shown in (F) are the means7SD (n¼3). **Po 0.01.
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640634
leukocyte adhesion to TNF-a-stimulated HUVECs. Keap1 knock-down effectively suppressed TNF-a-stimulated increase in theadhesion of monocytes to HUVECs, and this suppressive effectwas significantly reversed by treatment with SnPP and BSO, butnot with dicumarol (Fig. 3E and F). Taken together these resultsindicate that Keap1 knockdown may suppress vascular inflam-matory responses and potentially vascular disorders.
Keap1 knockdown suppresses TNF-a-induced NF-kB activation
The nuclear transcription factor NF-kB is critically involved inthe expression of the inflammation-associated genes, includingICAM-1 and VCAM-1 [17]. We investigated the effects of Keap1siRNA on the transcriptional activity of both gene promoterconstructs containing the proximal NF-kB-binding sites locatedat �200 bp (ICAM-1) and at 65 and 75 bp (VCAM-1) upstream oftheir transcription start sites [18]. Stimulation of HUVECs withTNF-a resulted in significant increases in both promoter activities,which were suppressed by Keap1 knockdown (Fig. 4A). We nextperformed a ChIP assay to determine the involvement of NF-kB inthe activation of both promoters. Binding of NF-kB to bothpromoters was significantly increased in TNF-a-stimulated cellscompared with control, and these interactions were inhibited byKeap1 knockdown (Fig. 4B). Moreover, these suppressive effectswere significantly reversed by treatment with SnPP and BSO, butnot with dicumarol (Fig. 4A and B). Similar inhibitory effects ofSnPP or BSO on NF-kB reporter activity were also observed inTNF-a-stimulated HUVECs after transfection with Keap1 siRNA(Fig. 4C). Furthermore, Keap1 knockdown inhibited nuclear
translocation of the NF-kB subunit p65 in TNF-a-stimulatedHUVECs, as confirmed by Western blotting and confocal micro-scopy (Fig. 4D and E). EMSA further demonstrated that Keap1knockdown suppressed NF-kB–DNA binding activity in thenuclear extract from HUVECs treated with TNF-a. The specificinteraction between DNA and NF-kB was demonstrated by acompetition assay with cold probe and a supershift experimentusing a NF-kB p65 antibody (Fig. 4F). These inhibitory effects ofKeap1 siRNA on TNF-a-induced NF-kB activation were signifi-cantly reversed by treatment with SnPP and BSO, but not withdicumarol (Fig. 4D–F). These results suggest that Keap1 knock-down inhibits the expression of ICAM-1 and VCAM-1 by suppres-sing nuclear translocation and activation of NF-kB via theinduction of HO-1 and GCL expression.
Keap1 knockdown inhibits TNF-a-induced NF-kB signaling pathway
The IkBa degradation is required for NF-kB nuclear migration[19]. Western blot analysis revealed that Keap1 knockdownsuppressed TNF-a-induced IkBa phosphorylation and degrada-tion, and these effects were reversed by treatment with SnPP andBSO, but not with dicumarol (Fig. 5A). Because phosphorylation ofIkBa is largely dependent on IKK activity, which is regulated bythe upstream signal mediators NIK and Akt [20–22], we examinedthe effect of Keap1 siRNA on phosphorylation-dependent activa-tion of NIK, Akt, and IKK. Keap1 knockdown effectively inhibitedTNF-a-induced increases in the phosphorylation of NIK, Akt, andIKKa/b at S176/177 and S180/181, and these inhibitory effectswere significantly reversed by treatment with SnPP and BSO, but
TNF-αsiRNA
Inhibitor
ICAM-1
VCAM-1
Input
Input
---
+--
++-
++
++
++
ICAM-1 VCAM-1
Lu
c a
cti
vit
y (f
old
)
TNF-αsiRNA
Inhibitor
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--
+
--
+
+-
+
+
+
+
5
3
2
1
0
4
+
+
+--
++-
4
3
2
1
0
Lu
c a
cti
vit
y (
fold
)
---
++
++
++
******
*****
---
+--
+
+-
+
+
+
+
p6
5P
AR
PNF
p6
5A
cti
nCF
TNF-αsiRNA
Inhibitor
CP
Ab
---
+--
++-
++
++
+-
-
+--
NF-κB
Supershift
TNF-αsiRNA
Inhibitor
p6
5D
AP
I---
+--
+
+-
+
+BSO
+
+SnPP
Me
rge
SnPP
BSO
DC
SnPP
BSO DC
SnPP
BSO
DC
SnPP
BSO SnPP
BSO
Fig. 4. Keap1 knockdown suppresses TNF-a-induced NF-kB activation. (A) HUVECs were cotransfected with 100 nM Keap1 siRNA and 1 mg/ml expression vector
containing ICAM-1 or VCAM-1 promoters for 30 h. Cells were treated with TNF-a in the presence or absence of chemical inhibitors for 12 h, and luciferase activity was
measured using a luminometer. Data shown are the means7SD (n¼4). *Po0.05 and **Po0.01. (B) Cells transfected with Keap1 siRNA were treated with TNF-a for 1 h in
the presence or absence of the enzyme inhibitors. ChIP analysis was performed as shown in Fig. 1E using primer pairs designed for ICAM-1 and VCAM-1 promoters.
(C) Cells were cotransfected with Keap1 siRNA and pGL3-NF-kB-Luc and treated as described for (A). Luciferase activity was measured by a luminometer. Data shown are
the means7SD (n¼3). **Po0.01. (D–F) Keap1 knockdown cells were treated with TNF-a for 1 h after pretreatment with SnPP and BSO for 1 h. Nuclear translocation of
NF-kB was determined (D) in cytosolic (CF) and nuclear fraction (NF) by Western blotting as well as (E) in intact cells by immunohistochemistry. (F) Nuclear NF-kB–DNA
binding activity was determined in the presence or absence of cold probe (CP) or antibody for NF-kB p65 (Ab) by EMSA.
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640 635
not with dicumarol (Fig. 5B). ROS are important second messengersfor the regulation of NF-kB activation, by activating its upstreamsignal modulators, including Akt, NIK, and IKK [14,20,22]. Conse-quently, we sought to determine the effects of Keap1 knockdownon the TNF-a-induced ROS production. Keap1 knockdown effec-tively inhibited TNF-a-induced intracellular ROS accumulation,and this inhibitory effect was reversed by treatment with SnPPand BSO, but not with dicumarol (Fig. 5C and D). Moreover, thesuppressive effect of Keap1 knockdown on TNF-a-induced NF-kBreporter activity was reduced by the addition of the hemedegradation product CORM-2 and the antioxidant N-acetylcysteine(Fig. 5E). These results indicate that Keap1 knockdown blocked theTNF-a-induced NF-kB pathway in endothelial cells through inhibi-tion of intracellular ROS accumulation by inducing HO-1 and GCLexpression.
Transfection with siRNA for Prx1, but not GSTM1, attenuates Keap1
knockdown-mediated suppression of adhesion molecule expression
Because Keap1 knockdown elevated the expression of Prx1 andGSTM1 (Fig. 2), we further examined the roles of these enzymesand proteins on TNF-a-induced adhesion molecule expression inHUVECs transfected with Keap1 siRNA. Keap1 knockdown resultedin increases in Prx1 and GSTM1 expression, which were suppressedby transfection with siRNAs for Prx1 and GSTM1 (Fig. 6A). Specificknockdown of Prx1, but not GSTM1, reduced the suppressive effectof Keap1 knockdown on TNF-a-mediated increases in ICAM-1and VCAM-1 expression, monocyte adhesion, and intracellularROS accumulation (Fig. 6B–D). Moreover, Prx1 knockdown atte-nuated Keap1 siRNA-mediated nuclear translocation of NF-kB andNF-kB-Luc reporter activity in HUVECs (Fig. 6E and F). These
results suggest that induction of Prx1, but not GSTM1, by Keap1knockdown also plays an important role in the regulation of NF-kBactivation and inflammatory gene expression.
Keap1 knockdown inhibits LPS-induced inflammatory response in
macrophages
Because NF-kB is also a key transcription factor for elicitingimmune responses to LPS [23], we next investigated whetherKeap1 knockdown regulates the production of cytotoxic media-tors, such as NO and TNF-a, which are responsible for septicshock. Transfection of RAW264.7 cells with Keap1-specific siRNAinhibited the levels of Keap1 mRNA and protein, leading to theinduction of HO-1, GCL, and Prx1 (Fig. 7A). Furthermore, transfec-tion with Keap1 siRNA suppressed the mRNA and protein levels ofiNOS and TNF-a in LPS-stimulated RAW264.7 cells (Fig. 7B).Under the same experimental conditions, Keap1 knockdowninhibited LPS-induced secretion of NO and TNF-a into the culturemedium of LPS-stimulated RAW264.7 cells (Fig. 7C and D). Thesedata indicate that Keap1 knockdown suppressed LPS-induced NOand TNF-a production in macrophages via the induction of HO-1,GCL, and Prx1.
Keap1 knockdown inhibits LPS-induced in vivo inflammation, tissue
damage, and lethality
To further investigate a role for Keap1 knockdown in in vivo
inflammatory processes, we determined the expression levels ofinflammation-associated genes in liver and lung tissues from mice,which were treated ip with LPS after intravenous injection withmixture of in vivo-jetPEI reagent and Keap1 siRNA. Administration
TNF- αsiRNA
Inhibitor
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S l
ev
el
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ld)
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0TNF-αsiRNA
Inhibitor
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uc a
cti
vit
y (
fold
)
p-IκBα
Actin
IκBα
IκBα
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+
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G132
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Inhibitor
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Inhibitor
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p-NIK
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p-Akt
p-IKKαβ(S176/177)
p-IKKαβ(S180/181)
**** **
SnPP
BSO
DC
SnPP
BSO
DC
SnPP
BSO
DC
NAC
CO
Fig. 5. Keap1 knockdown decreases TNF-a-induced NF-kB signaling pathway. (A and D) HUVECs transfected with Keap1 siRNA were allowed to recover in fresh medium
for 30 h and pretreated with 20 mM chemical inhibitors and 5 mM MG132 for 1 h, followed by stimulation with TNF-a for 30 min. (A) IkBa phosphorylation and
degradation and (B) phosphorylation of NIK, Akt, and IKKa/b were determined by Western blot analysis. (C and D) Under the same experimental conditions, intracellular
ROS accumulation was determined by confocal microscopy using DCFH2-DA. (a) Control; (b) TNF-a; (c) siRNAþTNF-a; (d) siRNAþTNF-aþSnPP; (e) siRNAþTNF-aþBSO;
(f) siRNAþTNF-aþdicumarol (DC). (E) Cells were cotransfected with Keap1 siRNA and pGL3-NF-kB-Luc for 30 h and treated with TNF-a in the presence or absence of
100 mM CORM-2 (CO) or 1 mM N-acetylcysteine (NAC) for 12 h. Luciferase activity was measured by a luminometer. Data shown in (D and E) are the means7SD (n¼3).
**Po0.01.
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640636
of Keap1 siRNA resulted in the reduction of Keap1 expression;elevation of HO-1, GCL, and Prx1 expression; and suppression ofICAM-1, VCAM-1, TNF-a, and iNOS expression in the liver and lungof LPS-treated mice (Fig. 8A). In addition, in vivo Keap1 knockdownsignificantly suppressed LPS-mediated increases in serum levels ofNOx and TNF-a (Fig. 8B and C). We next examined the functionalrole of Keap1 in LPS-mediated tissue damage in a mouse model.Administration with Keap1 siRNA prominently inhibited LPS-induced tissue injuries of liver and lung, showing distinct injuryand immune cell infiltration (Fig. 8D). Furthermore, administrationof LPS (35 mg/kg) resulted in a 24-h mortality rate of 100% in mice;however, preinjection with Keap1 siRNA significantly reduced LPS-induced mice lethality (Fig. 8E). These results provide evidence forthe protective effects of Keap1 knockdown on endotoxic tissueinjury and lethality by inhibiting inflammatory and cytotoxic geneexpression.
Discussion
This study was undertaken to elucidate the functional role ofNrf2-dependent phase II gene products in NF-kB activation andinflammatory responses in vitro and in vivo using Keap1-specificsiRNA and phase II enzyme inhibitors. We found that specificknockdown of Keap1 effectively induced Nrf2 activation and ARE/EpRE-dependent expression of phase II genes (HO-1, GCL, andPrx1), which were highly correlated with suppression of TNF-
a-induced ROS accumulation and NF-kB-mediated adhesionmolecule expression in HUVECs. Moreover, Keap1 knockdownsuppressed LPS-mediated iNOS and TNF-a expression in culturedmacrophages and in a mouse model, as well as protecting micefrom LPS-induced lethality. These results indicate that Nrf2 activa-tion prevents NF-kB-mediated vascular inflammation and endo-toxic shock by suppressing NF-kB-mediated inflammatory geneexpression via upregulation of Nrf2-mediated antioxidant genes.
Keap1 contains a number of redox-sensitive cysteine residues[24]. Of them, three residues, C151, C273, and C288, are critical tothe function of Keap1, which is a substrate adaptor for the Keap1–Cul3–E3 ubiquitin ligase complex. Under normal conditions, thiscomplex induces the ubiquitination of Nrf2 and its subsequentdegradation. However, modification of these cysteine residues byoxidants, heavy metals, and alkylating agents is considered toimpair the structural integrity of the Keap1–Cul3–E3 ligasecomplex, resulting in a decline in ubiquitination activity, therebyfacilitating accumulation of Nrf2 in the cytosol. The liberated andstabilized Nrf2 translocates into the nucleus and activates tran-scription of its downstream target genes by binding to ARE/EpREpresent in the promoters of antioxidant and cytoprotective phaseII detoxifying genes.
Nrf2 is known to induce phase II genes, such as HO-1, Prx1,GCL, GST, and NQO1. HO-1 breaks heme down to carbon monoxide(CO), ferrous iron, and biliverdin/bilirubin. These by-products func-tion as antioxidants under physiological and pathological condi-tions [25–27]. Thus, induction of HO-1 is directly associated with
CRL
TNF-α +siR-Keap1
TNF-α +siR-Keap1
+ siR-Prx1
Anti-p65
DAPI
Merge
siR-Keap1siR-Prx1
siR-GSTM1
ICAM-1
VCAM-1
Actin
TNF-αsiR-Keap1
siR-Prx1siR-GSTM1
Lu
c a
cti
vit
y (
fold
)
---
+--
++-
+++
7
3
2
1
0TNF-α
siR-Keap1siR-Prx1
5
4
6
----
+---
++--
+++-
0
Mo
no
cyte
ad
he
sio
n
(Fo
lds
)
10
8
6
4
2
TNF-αsiR-Keap1
siR-Prx1siR-GSTM1
----
+---
++--
+++-
0
RO
S (
Fo
lds
)
25
20
15
10
5
TNF-αsiR-Keap1
siR-Prx1siR-GSTM1
Keap1
HO-1
Actin
Prx1
---
+--
++-
+-+
GAPDH
GSTM1
----
+---
++--
+++-
++-+
++-+
++-+
** ** ** **
** **TNF-α
Fig. 6. Keap1 knockdown induces Prx1, which is responsible for regulation for NF-kB-dependent adhesion molecule expression. (A) The mRNA and protein levels of Keap1,
Prx1, and GSTM1 were determined in HUVECs transfected with 100 nM Keap1 siRNA for 30 h by Western blotting and RT-PCR. (B) Cells cotransfected with 100 nM siRNAs
for Keap1, Prx1, and GSTM1 were treated with TNF-a for 12 h. The protein levels of ICAM-1 and VCAM-1 were determined by Western blotting. (C) Monocyte adhesion,
(D) intracellular levels of ROS, (E) nuclear translocation of NF-kB, and (F) NF-kB promoter activity were determined in HUVECs transfected with siRNA against Keap1, Prx1,
and GSTM1 as described for Figs. 3E, 5C, 4E, and, and 5E, respectively. Data shown in (C, D, and F) are the means7SD (n¼3). **Po0.01.
Keap1
HO-1
Actin
siRNA -
Keap1
HO-1
Actin
LPS
siRNA
-
-
+
-
+
+
-
+
3
2
1
0
TN
F-α
(ng
/ml)
**
LPS
siRNA
-
-
+
-
+
+
-
+
Nit
rite
(μM
)
20
15
10
5
0
**
Prx1
GCLC
GCLM
LPS
siRNA
iNOS
Actin
TNF-α
-
-
+
-
+
+
-
+
iNOS
Actin
TNF-α
Fig. 7. Keap1 knockdown inhibits inflammatory gene expression in LPS-activated RAW264.7 cells. (A) RAW264.7 cells were transfected with 50 and 100 nM Keap1 siRNA
for 30 h. The expression levels of Keap1 and some phase II genes were determined by Western blotting and RT-PCR. (B–D) Cells were transfected with 100 nM Keap1 siRNA
for 30 h and treated with LPS (1 mg/ml) for 12 h. (B) The expression levels of iNOS and TNF-a were determined by Western blotting and RT-PCR. The levels of (C) nitrite and
(D) TNF-a were determined by Griess reaction and ELISA, respectively. **Po0.01.
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640 637
cytoprotection and suppression of NF-kB-mediated inflammatorygene expression [28–30]. Prx1 is a thiol-containing small redoxprotein involved in the suppression of intracellular ROS accumula-tion [31]. GCL is a rate-limiting enzyme in cellular GSH synthesis,leading to an increase in cellular redox potential [32]. AlthoughNQO1 does not possess direct antioxidant activity, its majorfunction is to detoxify quinones and their derivatives, thus protect-ing cells from oxidative stress by removing compounds capable ofgenerating ROS [33]. GST family enzymes catalyze the conjugationof GSH to toxic compounds, resulting in more water-soluble and
less biologically active products that may easily be excreted [34];however, GSTM1, an isoform of the GST family, has been shown toinhibit intracellular accumulation of ROS [35]. Thus, phase II geneproducts regulate cellular redox potential by blocking intracellularROS accumulation through two distinct mechanisms, suppressionof ROS generation and elevation of ROS removal activity. We herefound that Keap1 knockdown effectively induces Nrf2-dependentphase II gene expression, subsequently resulting in inhibition ofTNF-a-induced intracellular accumulation of ROS, which is respon-sible for NF-kB activation (Figs. 2, 4, 5, and 6). Importantly, this
CTRL LPS+siRNA
Liv
er
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ng
0 6 12 18 24 30 36
Time (h)
0
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rviv
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siRNA + LPS
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siRNA
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l)
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siRNA
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siRNA
Actin
ICAM-1
iNOS
TNF-α
Keap1
HO-1
VCAM-1
Liver Lung
-
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-
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+
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+
+
Prx1
GCLC
GCLM
LPS
Fig. 8. Keap1 knockdown inhibits LPS-induced inflammation, tissue damage, and lethality in a mouse model. Mice were injected ip with LPS (35 mg/kg) after 24 h of
intravenous injection with a mixture of Keap1 siRNA (0.5 mg/kg) and in vivo-jetPEI reagent. (A) After 12 h, the expression levels of target genes were determined in the
tissues of liver and lung by Western blotting and RT-PCR. (B and C) The levels of NOx and TNF-a were determined in sera from mice treated with LPS for 12 h using
commercial assay kits. Data shown are the means7SD (n¼6). **Po0.01 versus LPS alone. (D) Thin sections of liver and lung from mice treated with LPS for 12 h were
stained with hematoxylin and eosin. Each section was examined under an optical microscope (original magnification �100). The photographs show representative results
from six mice per group. (E) Mouse mortality was determined every 6 h after injection of LPS. Each experimental group consisted of six mice.
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640638
suppressive effect was reduced by treatment with HO-1 and GCLinhibitors and Prx1 siRNA, but not with NQO1 inhibitor and GSTM1siRNA. These effects are likely to be associated with production ofCO, biliverdin/bilirubin, and iron from heme by HO-1 [25–27];de novo synthesis of GSH by GCL [32]; and expression of thiol-containing Prx1 [31]. These biological molecules are involved in theinhibition of NAD(P)H oxidase activity [36,37] and promotion ofROS-scavenging activity [38]. However, NQO1 and GST are notimplicated in the suppression of intracellular ROS accumulation inTNF-a-stimulated HUVECs. These findings suggest that induction ofthese phase II genes by Keap1 knockdown, with the exception ofNQO1 and GST, is responsible for regulating cellular redox potentialin TNF-a-activated endothelial cells.
Although high levels of ROS are toxic to cells, physiological levelsof ROS serve a signaling function to regulate various biologicalprocesses, such as cell proliferation and migration and gene expres-sion [39]. ROS produced by immune stimulants, including TNF-aand LPS, play a critical role in inflammatory gene expression viaactivation of the NF-kB signaling pathway. Our previous studydemonstrated that the antioxidant lutein inhibits NF-kB activationand inflammatory gene expression in LPS-stimulated macrophagesby suppressing redox-sensitive activation of the PTEN/Akt and NIK-dependent pathways through removal of intracellular ROS [14].Both NIK and Akt are involved in NF-kB activation in TNF-a- andLPS-stimulated cells through the phosphorylation of IKKa. Thispromotes IKKb phosphorylation in a reciprocal activation mode,demonstrating an involvement of Akt and NIK in the canonical NF-kB activation pathway [22,40,41]. These observations indicate that
ROS are involved in the activation of the canonical pathway via areciprocal cross talk between IKKa and IKKb by activating Akt andNIK. We here found that Keap1 knockdown promoted Nrf2-depen-dent antioxidant gene expression and removal of intracellular ROS,which is responsible for NF-kB activation and inflammatory geneexpression (Figs. 2 and 5). These findings indicate that Nrf2 activa-tion exerts anti-inflammatory effects through inhibition of NF-kBactivation by inducing phase II antioxidant gene expression.
Endothelial cells play an important role in host immune res-ponses for vascular homeostasis during inflammation and infection.Endothelial cells activated by immune stimulants upregulate theadhesion molecules ICAM-1 and VCAM-1, which are involved in themigration of peripheral blood leukocytes to the sites of inflamma-tion or infection [42]. Although leukocyte recruitment is importantfor eliminating invading pathogens, excessive leukocyte accumula-tion in the site of inflammation can lead to tissue damage [42]. Theexpression of ICAM-1 and VCAM-1 in endothelial cells can betightly regulated by NF-kB activation. Antioxidants have beenshown to inhibit NF-kB activation and block leukocyte infiltrationinto sites of inflammation by suppressing the expression of adhe-sion molecules, implicating endothelial cells as a target for regu-lating inflammatory processes [43]. We here found that Keap1knockdown attenuated monocyte adhesion to TNF-a-stimulatedHUVECs by inhibiting NF-kB-dependent expression of ICAM-1 andVCAM-1 (Fig. 3), which participate in the pathogenesis of sepsis andatherosclerosis. These inhibitory effects were highly correlated withthe expression levels of HO-1, GCL, and Prx1 and were suppressedby selective inhibition of these enzymes/proteins (Figs. 3 and 6).
J.-H. Kim et al. / Free Radical Biology and Medicine 53 (2012) 629–640 639
These results suggest that phase II gene products are involvedin inhibiting TNF-a-mediated expression of adhesion moleculesin HUVECs via suppression of ROS-mediated NF-kB activation.Moreover, systemic delivery of Keap1 siRNA decreased the expres-sion of adhesion molecules and infiltration of leukocytes into liverand lung, which are major organs of septic injury, in an endotoxe-mic mouse model (Fig. 8). These findings indicate that Keap1 siRNAcan prevent endotoxic shock by suppressing NF-kB-dependentadhesion molecule expression.
LPS, a prime initiator of septic shock, activates macrophages andendothelial cells and upregulates a variety of NF-kB-dependentinflammatory genes, leading to production of the cytotoxic mediatorsTNF-a, interleukin-1b, and NO. These mediators are known toincrease tissue damage and lethality [14,44]. Thus, NF-kB inhibitorshave clinical potential for controlling various inflammation-asso-ciated human diseases [5,6]. We found that Keap1 knockdownsuppressed the expression of NF-kB-dependent inflammatory genes,TNF-a and iNOS, in LPS-stimulated RAW264.7 cells (Fig. 7). On theother hand, delivery of Keap1 siRNA significantly induced Nrf2-dependent expression of phase II genes and suppressed the expres-sion of TNF-a, iNOS, and adhesion molecules in LPS-administeredmice (Fig. 8). Furthermore, in vivo Keap1 knockdown reduced organinjuries and lethality in a mouse model of endotoxic shock. Thesefindings suggest that siRNA targeting Keap1 can be used as atherapeutic agent for treating various inflammatory diseases, includ-ing septic or endotoxic shock.
Recent genome-wide analysis demonstrated that Nrf2 regulateshundreds of genes that are involved in the cytoprotective responseagainst oxidative stress [45]. Although we did not determine theexpression levels of all these genes in this study, it is feasible thatKeap1 knockdown could induce the expression of many otherantioxidant-related genes. Although not shown, Keap1 knockdowndid not significantly increase other antioxidant enzymes, suchas superoxide dismutase-1, catalase, and peroxidase-1, as deter-mined by RT-PCR. Therefore, we excluded the notion that theseenzymes could not contribute to the regulation of NF-kB-mediatedinflammatory gene expression under our experimental conditions.Anti-inflammatory effects induced by Keap1 knockdown or Nrf2-activating compounds may be elicited by cross talk of multiplepathways or synergistic interaction among Nrf2-targeting geneproducts. Therefore, the beneficial effect of Keap1 knockdowncannot be fully explained by upregulation of three antioxidantgene products, HO-1, GCL, and Prx1; however, these enzymes/proteins may play an important role in the immune-suppressiveeffect of siRNA-based Keap1 knockdown.
Taken together, our data show that siRNA-mediated knock-down of Keap1 increased Nrf2-dependent expression of the phaseII genes HO-1, GCL, and Prx1, which are implicated in thesuppression of intracellular ROS accumulation in immune-acti-vated cells. This suppressive effect is directly associated withinhibition of the signal mediators Akt, NIK, and IKK, which areupstream of NF-kB activation, resulting in the suppression ofinflammation-associated gene expression in TNF-a-stimulatedendothelial cells and LPS-treated macrophages. Moreover, sys-temic delivery of Keap1 siRNA protected mice from LPS-inducedorgan injury and lethality. These findings provide a rationale fornovel therapeutic approaches utilizing Keap1-specific siRNA forhuman inflammatory diseases, such as sepsis and atherosclerosis.
Acknowledgments
This work was supported by a Korea Science and EngineeringFoundation grant funded by the Korean government (MOST)(M10642140004-06N4214-0040).
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