Molecular Immunology 56 (2013) 113122

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Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm

Proteomic changes induced by histone demethylase JMJD3 in TNF alpha-treated

human monocytic (THP-1) cells
Amitabh Das 1 , Nando Dulal Das 1 , Kyoung Hwa Jung, Ji Hyun Park, Hyung Tae Lee, DalMuri Han,
Mi Ran Choi, Sung Chul Kang, Young Gyu Chai
Department of Molecular & Life Science, Hanyang University, Ansan, 426-791, Republic of Korea

a r t i c l e

i n f o

Article history:
Received 5 October 2012
Received in revised form 16 March 2013
Accepted 23 April 2013
Available online 25 May 2013
Keywords:
Inammation
Ingenuity Pathway Analysis (IPA)
JMJD3
MALDI-TOF MS
NF-B
THP-1 cell

a b s t r a c t
JMJD3, a Jumonji C family histone demethylase, plays an important role in the regulation of inammation induced by the transcription factor nuclear factor-kappa B (NF-B) in response to various stimuli.
JMJD3 is a histone-3 lysine-27 trimethylation (H3K27me3) demethylase, a histone mark associated
with transcriptional repression and activation of a diverse set of genes. The present study assessed
stable JMJD3 knockdown (KD)-dependent proteomic proling in human leukemia monocyte (THP-1)
cells to analyze the JMJD3-mediated differential changes of marker expression in inammatory cells.
To analyze the protein expression prole of tumor necrosis factor-alpha (TNF-)-stimulated JMJD3-kd
THP-1 cells, we employed matrix-assisted-laser-desorption/ionization-time-of-ight mass spectrometry
(MALDI-TOF MS). Additionally, Ingenuity Pathways Analysis (IPA) was applied to establish the molecular
networks. A comparative proteomic prole was determined in TNF--treated both of JMJD3-kd THP1 cells and THP-1 scrambled (sc) cells. The expression of tripartite motif protein (TRIM5), glutathione
peroxidase (GPx), glia maturation factor- (GMFG), caspase recruitment domain family, member 14
(CARMA2), and dUTP pyrophosphatase were signicantly down-regulated, whereas heat shock protein
beta-1 (HspB1) and prohibition were signicantly up-regulated in JMJD3-kd THP-1 cells. The molecular
and signaling networks of the differentially expressed proteins in JMJD3-kd THP-1 cells were determined
by IPA. The molecular network signatures and functional proteomics obtained in this study may facilitate
the suppression of different key inammatory regulators through JMJD3-attenuation, which would be
crucial to evaluate potential therapeutic targets and to elucidate the molecular mechanism of JMJD3-kd
dependent effects in THP-1 cells.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Histone modications regulate gene transcription by affecting chromatin compaction, DNA accessibility, and recruitment
of transcription machinery and regulators. Various modications
such as methylation, phosphorylation, acetylation, and ubiquitination occur at residues in the amino- and carboxyl-terminal
tails of core histones in several different ways (Cheung et al.,
2000; Berger, 2007). Among these, modications by histone acetylation and methylation are the most common. Regulation of a

Corresponding author at: Department of Molecular & Life Science, Hanyang University, 1271 Sa 3-dong, Ansan, Gyeonggi-do 426-791, Republic of Korea.
Tel.: +82 31 400 5513; fax: +82 31 406 6316.
E-mail addresses: amitabhdas.kn@gmail.com (A. Das), nando.hu@gmail.com
(N.D. Das), khjung2@gmail.com (K.H. Jung), b88424@gmail.com (J.H. Park),
photod@naver.com (H.T. Lee), holystom@gmail.com (D. Han),
miran choi@hotmail.com (M.R. Choi), gujiju11@gmail.com (S.C. Kang),
ygchai@hanyang.ac.kr (Y.G. Chai).
1
These authors contributed equally to this work.
0161-5890/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.molimm.2013.04.013

variety of nuclear processes dedicated to the maintenance of active

and silent states of gene expression require histone methylation,
which is essential for cellular regulation, homeostasis, and fate
determination (Cloos et al., 2008). Multiple lysine residues on
histones, including H3K4, H3K9, H3K27, and H3K36, are methylated by histone methyltransferases and demethylated by histone
demethylases. A large number of demethylases with the ability to
demethylate specic histone lysine and arginine residues via amine
oxidation, hydroxylation, or deamination have been discovered
(Shi and Whetstine, 2007).
JMJD3, a JmjC family histone demethylase, erases H3K27me3,
a histone mark associated with transcriptional repression and
involved in lineage determination. H3K27me3 is crucial for gene
silencing. To allow commitment and differentiation to various
cell lineages, this silencing state can be altered through the
activity of the JMJD3 demethylase. Importantly, NF-B-induced
JMJD3 up-regulation relates inammation to the control of
histone modication involved in lineage determination, differentiation, and tissue homeostasis, which may provide a connection
between chronic inammation and the associated alterations of

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A. Das et al. / Molecular Immunology 56 (2013) 113122

Table 1
Up-regulated proteins in 2 h TNF-treated JMJD3-kd THP-1 cells.
Protein name

Spot no.

THP1 sc

THP1 JMJD3kd

Ratio

Estd Z

Access number

FA81A HUMAN RecName: Full = Protein

FAM81A
WDR67 protein
Hypothetical protein
hCG2010950
Keratin 10
Unnamed protein product
Heat shock protein beta-1
Deoxyribonucleoside 5 -monophosphate
N-glycosidase isoform 1
Heat shock 27 kDa protein 1
Beta actin variant
Prohibitin
Folate binding protein
Unnamed protein product
Nucleoporin like 2, isoform CRA d
Tat binding protein 1,
TBP-1 = transcriptional activator [human,
Peptide, 439 aa]
Zinc nger protein 716
Interleukin 3 receptor, alpha
Protein disulde-isomerase A3 precursor
Unnamed protein product

2524

64,919

64,919

2.43

gi74730462

4423
1505
1312
4424
1722
5302
2217

1
1
1
1
1
1
28

17,182
8322
3850
2400
528
244
2235

17,182
8322
3850
2400
528
244
79.82

1.14
2.43
1.1
2.26
2.43
2.43
0.94

gi20072649
gi6599170
gi119578959
gi186629
gi194390376
gi4504517
gi5454002

4313
5510
5409
2401
2525
6615
3623

554
8718
640
45,765
2402
110
3335

11,027
81,515
5129
116,687
15,816
622
14,581

19.9
9.35
8.01
7.4
6.58
5.65
4.37

1.32
2.43
2.43
0.23
1.43
1.33
2.43

gi15126735
gi62897625
gi46360168
gi2565194
gi16550217
gi119614184
gi263098

5728
2611
6714
6510

1278
19,382
36,596
9660

4335
64,919
95,179
18,098

3.39
3.349
2.6
1.87

1.88
1.43
2.43
2.43

gi226529227
gi57163101
gi21361657
gi194376310

differentiation (De Santa et al., 2009). As histone methylation is a

reversible dynamic process mediated by methyltransferases and
demethylases, whether and how these processes are regulated in
acute to chronic inammatory responses has yet to be elucidated.
H3K27 methylation, marked as a repressive histone modication,
acts by recruiting polycomb complexes that identify a checkpoint
regulator of inducible gene expression. It is reported that JMJD3
is up-regulated after LPS stimulation and can induce expression of
a subset of genes, such as BMP2 and HOX genes, through H3K27
demethylase function (De Santa et al., 2007). Interestingly, this
repressive histone modication by JMJD3 could be one of the
targets of the anti-inammatory signaling pathway and gene specic inhibition of inammatory responses (Medzhitov and Horng,
2009).
Several histone modications have been reported by the differential regulation of JMJD3 and NF-B targeted genes. However,
very little work has been done to determine the importance
of proteomic proling on JMJD3 in regulating NF-B activity
and other cellular processes. Because of JMJD3 induction in
macrophages (De Santa et al., 2009) and monocytes (Das et al.,
2012a,b) exposed to bacterial products and inammatory cytokines
such as TNF-, we chose THP-1 cells to study the involvement
ofJMJD3 in inammatory conditions to determine whether the
proteomic changes affect different signaling pathways such as, NFB signaling and other cytokine signaling pathways. Our primary
objective was to compare the protein expression prole of TNF-treated JMJD3-attenuated THP-1 (JMJD3-kd THP-1) cells with
that of THP1 sc cells using two-dimensional polyacrylamide gel
electrophoresis (2D)/matrix-assisted-laser-desorption/ionizationtime-of-ight mass spectrometry (MALDI-TOF MS). Recently,
proteomic techniques have emerged as a powerful tool to
relate broad-spectrum protein expression with specic cellular
responses. In our study, a comparative protein expression signature was established for TNF--treated both of JMJD3-kd THP-1
cells and THP1 sc to identify novel molecular targets of the JMJD3kd THP-1 cells in cytokine-stimulated inammatory conditions. In
particular, we detected differential expression of proteins including tripartite motif protein (TRIM5), glutathione peroxidase (GPx),
Rho GDP-dissociation inhibitor 1 (RHOGDI1), nuclear distribution gene C (NudC), nudix hydrolase (NUDT5), glia maturation
factor- (GMFG), caspase recruitment domain family, member 14
(CARMA2), dUTP pyrophosphatase, WDR67, heat shock protein

beta-1 (HspB1), prohibition and folate binding protein (FBP) in TNF-treated JMJD3-kd THP-1 cells. Overall, the effects suggest that
JMJD3 might be a therapeutic target with possible research and
clinical value. In addition, the signaling pathways and molecular
networks induced by 2 h of TNF- treatment of JMJD3-kd THP-1
cells were determined by Ingenuity Pathway Analysis (IPA). These
pathways have the potential to dene the molecular targets associated with JMJD3-kd THP-1 cells. Notably, the molecular network
characterized by IPA analysis showed that TNF--treated JMJD3-kd
THP-1cells up-regulated heat shock protein and down-regulated
TRIM5, protein disulde isomerase, NudC for the involvement of
interleukin 15, immunoglobulin, and hepatocyte nuclear factor 4,
alpha (HNF4), which is due to the JMJD3-attenuation that could
affect the inammatory condition in THP-1 cells. The top ve
diseases and disorders associated with down-regulated genes in
TNF--treated JMJD3-kd THP-1 cells, as determined by IPA analysis, were inammatory disease, respiratory disease, immunological
disease, genetic disorder and hematological disease.
2. Materials and methods
2.1. Reagents
Cell culture medium RPMI 1640, fetal bovine serum (FBS) and
penicillinstreptomycin (PS) liquid were purchased from Invitrogen (USA). TNF- was purchased from SigmaAldrich (USA).
The lentiviral vector-based JMJD3 short hairpin RNA (shRNA) and
scrambled shRNA were synthesized for this work (available on
request).
2.2. Establishment of JMJD3-kd THP-1 cells and treatments
Establishment of scrambled shRNA-transfected, THP1 sc and
JMJD3 shRNA-transfected, and JMJD3-kd THP-1 cells was performed as described in our previous paper (Das et al., 2012a,b).
Human monocytic THP-1 cells were originally obtained from the
American Type Culture Collection (Manassas, VA). Cell cultures
were maintained at sub-conuence in a 95% air, 5% CO2 humidied atmosphere at 37 C using RPMI 1640 medium supplemented
with 10% heat-inactivated FBS, 1% PS, glutamine, HEPES and 50 M
-mercaptoethanol (Shanmugam et al., 2003). Both THP-1 sc and

A. Das et al. / Molecular Immunology 56 (2013) 113122

JMJD3-kd THP-1 cells were treated with TNF- (10 ng/ml) for a
period of 2 h under normal culture conditions.
2.3. Protein sample preparation
Proteins were extracted in lysis buffer (9.5 M urea, 2.5%
3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate
(CHAPS), 40 mM dithiothreitol (DTT), 0.12% carrier ampholytes,
0.0012% bromophenol blue and solubilized by sonication on ice.
The protein samples were prepared as previously described (Jung
et al., 2010). The protein concentrations of 10-fold dilutions of the
samples were measured using the Bradford assay (SigmaAldrich
Inc., USA), with BSA (2 mg/ml; Pierce Biotechnology, Inc., Rockford,
IL) as a standard.
2.4. Two-dimensional gel electrophoresis (2-DE) and in-gel
digestion
Protein samples were subjected to two-dimensional gel electrophoresis (2-DE) using our previously published methods (Jung
et al., 2010). Analysis of the 2-DE digitized images was carried
out using PDQuest software (version 7.3, Bio-Rad Laboratories,
Hercules, CA) to quantify the spot densities. Three independent
2-DE analyses were performed with similar gel images being
obtained from each individual experiment. Proteins that showed
changes more than 2-fold from three independent experiments
were selected for further characterization. The relative spot volume of each protein compared to the normalized protein volume
was used to determine the relative expression level of individual
protein spots. Enzymatic digestion of the protein spots was carried
out in-gel as described previously (Das et al., 2012a,b; Shevchenko
et al., 1996), using modied porcine trypsin (Promega, Madison,
WI).
2.5. Matrix-assisted laser desorption/ionization-time-of-ight
mass spectrometry and database analysis
Peptide analysis was performed using an Ultraex 2 matrixassisted-laser-desorption/ionization (MALDI)-time-of-ight (TOF)
mass spectrometer (Bruker Daltonics, Bremen, Germany). In
brief, peptides were evaporated using an N2 laser (337 nm) in
a delayed-extraction approach and were accelerated with a
20-kV injection pulse for duration of ight analysis. Each spectrum represented the cumulative average of 300 laser shots.
The ProFound search program was developed at Rockefeller
University (http://prowl.rockefeller.edu/prowl-cgi/profound.exe)
and was used for protein identication employing a Bayesian
algorithm based on peptide mass ngerprinting data. The
presence of signal peptides was considered when such
information was available in the corresponding National
Center for Biotechnology Information GenPept format atle
(www.ncbi.nlm.nih.gov/Entrez/batch.html). The following input
parameters were applied: mass tolerance-1, iodoacetamide
processing for amino acid residues. The spectra were internally
calibrated using trypsin autodigestion ion peaks (m/z 842.510 and
2211.1046).
2.6. Western blot analysis
It was performed according to standard procedures (Das et al.,
2010). Briey, whole cell lysates (20 g) were resolved by electrophoresis on 12% polyacrylamide gels. Proteins were then
transferred to polyvinylidene diuoride (PVDF) membranes (Schleicher & Schuell Bioscience, Inc., Keene, NH) by electroblotting
using the semi-dry method. The membranes were blocked with 5%
skimmed milk in 1% TBS-Tween for 1 h and incubated overnight

115

with primary antibodies at 4 C. After washing, the membranes

were incubated for 1 h with the secondary antibody. Blots were
visualized with enhanced chemiluminescence (ECLTM Plus Western
Blotting Detection Kit; GE Healthcare, Piscataway, NJ). The antibodies used were JMJD3 (sc-130157), NUDC (sc-160616), HspB1
(sc-69702), -actin (sc-8432) from Santa Cruz Biotechnology and
TRIM5 (Ab-24181), FBP (Ab-3361) from Abcam.
2.7. Quantitative real-time RT-PCR
Real-time RT-PCR was performed using the SYBR Green PCR
Master Mix (Takara Bio Inc., Shiga, Japan) and the 7500 fast real time
PCR system (Applied Biosystems, Foster City, CA). Primer design
was performed using Primer Express (Applied Biosystems, Foster City, CA), and all primers are listed in Table S1. The detailed
methods were described previously (Baik et al., 2005; Das et al.,
2010). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was
used as an internal control. All data were analyzed using the 2CT
method.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molimm.
2013.04.013.
2.8. Canonical pathway analysis of datasets
IPA (Ingenuity Systems [www.ingenuity.com], Mountain
View, CA, USA) was conducted to determine the most signicant
canonical pathways in the datasets. Detailed methods for this procedure have been described previously (Li et al., 2007). In brief,
genes from the dataset were considered for literature review. The
signicance of the association between the dataset and the canonical pathway was measured in two ways: (1) the ratio of the number
of genes from the dataset that map to a given canonical pathway
was divided by the total number of genes that map to the same
canonical pathway; and (2) Fishers exact test was used to calculate a P-value to determine the probability that the association
between the genes in the dataset and the canonical pathway could
be explained by chance alone. After the datasets were uploaded,
each gene identier was mapped to its corresponding gene object
in the IPA Knowledge Base, and these genes were overlaid onto a
global molecular network. Gene networks were then algorithmically generated based on their connectivity.
2.9. Graphical representation of networks/pathways
Genes or gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge
(line). All edges are supported by the reference literature and
canonical information stored in the IPA Knowledge Base. The intensity of the node color indicates the degree of up-regulation (red) or
down-regulation (green).
2.10. Statistical analysis
Statistical analysis was performed using SPSS 17.0 (SPSS Inc.,
IL, USA). Data were tested using a one-way ANOVA followed by
the Tukeys HSD post hoc test. *P values of <0.05 were considered
signicant.
3. Results and discussion
Both THP1 sc cells and JMJD3-kd THP-1 cells were treated with
TNF- (10 ng/ml) for 2 h, and their protein expression patterns
were analyzed using a 2-DE (pH 3.010) technique. The rst dimension IEF separation was performed on an IPG strip, and the second
dimension was performed subsequently on 1016% SDS-PAGE.

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A. Das et al. / Molecular Immunology 56 (2013) 113122

Table 2
Down-regulated proteins in 2 h TNF-treated JMJD3-kd THP-1 cells.
Protein name

Spot no.

Ratio

Estd Z

Access number

Proliferation-inducing protein 38, isoform

CRA b
Protein disulde isomerase-related protein 5
PPIL4 protein
Chain A, crystal structure of a soluble dimeric
form of oxidized Clic1
Elongation factor 1-delta isoform 4
NudC domain containing 3
Glutathione peroxidase
Chain A, human Dutp pyrophosphatase
complex with Dudp
P-450 IIA3 protein (1 is 3rd base in codon)
Tripartite motif protein TRIM5 isoform delta
Troponin T type 1 (skeletal, slow), isoform
CRA c
Vimentin variant 3
Fas-ligand associated factor 2
Nudix hydrolase NUDT5
AF351784 1 dopamine receptor interacting
protein
Chain A, semi-extended solution structure of
human myeloma immunoglobulin D
Protein disulde-isomerase precursor
Capping protein (actin lament) muscle Z-line,
beta
Procollagen-proline, 2-oxoglutarate
4-dioxygenase (proline 4-hydroxylase), beta
polypeptide, isoform CRA d
PRED65
GMFG protein
Rho GDP-dissociation inhibitor 1
6-Phosphogluconolactonase
Glutathione peroxidase
Caspase recruitment domain family, member
14, isoform CRA c
Calreticulin precursor
ST8 alpha-N-acetyl-neuraminide
alpha-2,8-sialyltransferase 3
Human glutamine-fructose-6- phosphate
transaminase 1 (Gfpt1) in complex with
fructose 6-phosphate
GRP78 precursor
Ribonuclease HI large subunit

2714

THP1 sc
34,849

THP1 JMJD3kd
1

1.4

gi119629097

3621
2527
3438

22,697
20,972
13,141

1
1
1

0
0
0

2.43
1.34
2.34

gi1710248
gi78395001
gi39654881

2423
3313
2218
5210

11,474
4962
3431
2927

1
1
1
1

0
0
0
0

2.43
0.89
2.43
1.49

gi194239729
gi13277564
gi577777
gi34810576

3720
3524
2320

1885
1017
635

1
1
1

0
0
0

1.91
2.43
0.85

gi825700
gi12407387
gi119592744

1612
1223
2422
4207

586
483
444
304

1
1
1
1

0
0
0
0

2.43
0.61
2.36
1.03

gi167887751
gi2731639
gi6694937
gi14194057

5410

262

0.71

gi82407638

5729
5406

24,112
5130

413
186

0.017
0.036

2.43
1.45

gi20070125
gi55664833

3520

21,999

947

0.043

1.42

gi119610102

5603
3113
3301
5303
6207
1610

14,808
950
15,874
775
2287
4829

1083
100
1872
159
539
705

0.073
0.105
0.117
0.205
0.235
0.145

0.89
2.13
1.5
2.43
2.43
0.25

gi7717243
gi34783447
gi4757768
gi6912586
gi4467837
gi119609994

1608
2528

3598
6687

705
2353

0.195
0.351

2.19
2.43

gi4757900
gi119583447

3437

6144

2162

0.351

0.9

gi209156383

3801
3439

14,008
3799

6238
2162

0.445
0.569

2.38
2.43

gi386758
gi3334761

Estd Z, estimated Z score as a measure of condence for profound-based identication

We used alkaline silver staining, which detected approximately

1800 200 spots per gel (Fig. S2). Gel images were analyzed using
PDQuest 7.3 image software, which permitted a comparison of
the expression patterns of proteins after TNF- treatment. The
differential spots were processed for MALDI-TOF-MS after in-gel
digestion using trypsin. Searching for selected differential spots
using the ProFound database, we were able to identify a total of 54
up- and down-regulated proteins. The 2 h TNF- exposure resulted
in the signicant down-regulation of the following proteins in
JMJD3-kd THP-1 cells compared with 2 h TNF- treated THP1 sc
cells: TRIM5, GPx, RHOGDI1, NudC, NUDT5, GMFG, CARMA2, and
dUTP pyrophosphatase were signicant (Table 2). In addition, a few
up-regulated proteins were detected, including WDR67, HspB1,
prohibitin, and FBP, all of which showed signicant differences
(Table 1) ().
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molimm.
2013.04.013.
TRIM5 was signicantly down-regulated in TNF--treated
JMJD3-kd THP-1 cells (Fig. 1A). TRIM5 is capable of activating
mitogen activated protein kinases (MAPK)- and NF-B-dependent
inammatory genes involved in the innate immune response similar to those activated by the gram-negative bacterial cell wall
component lipopolysaccharide (LPS) (Pertel et al., 2011) through

Toll-like receptor 4 (Brenchley et al., 2006). Further investigation

revealed that TRIM5 interacts with transforming growth factor
beta-activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB2),
and TAB3, components of the TAK1 kinase complex, in order to
activate MAPK and NF-B signaling pathways (Shi et al., 2008).
Regarding these ndings, down-regulation of TRIM5 can be a
potential target for the inhibition of MAPK- and NF-B-dependent
inammatory genes involved in the innate immune response in
TNF--treated JMJD3-kd THP-1 cells where JMJD3 attenuation can
control the TRIM5-mediated inammatory mechanism. Another
signicant down-regulated protein in TNF--treated JMJD3-kd
THP-1 cells was GPx. GPx families, which are widely expressed
in various tissues, are the most important reactive oxygen species
(ROS) scavengers. ROS, such as hydroxyl radical, hydrogen peroxide and superoxide radical, are well-known cytotoxic molecules
directly implicated in the etiology of a wide range of human
diseases, including cancer (Waris and Ahsan, 2006). Therefore,
down-regulation of GPx may have a possible role in preventing
cancer as well as other human diseases through induction of ROS
expression, as we also found down-regulation of GPx in TNF-treated JMJD3-attenuated human leukemia monocyte (THP-1)
cells. RHOGDI1 was also down-regulated in TNF--treated JMJD3kd THP-1 cells. The only known functions of RHOGDIs are their
interactions with and regulation of RHO GTPases. When RHOGDI1

A. Das et al. / Molecular Immunology 56 (2013) 113122

117

Fig. 1. Enhanced segments of the two-dimensional gel electrophoresis gel derived from different treated samples: (A and B) both THP1 sc cells (left panels) and JMJD3-kd
THP-1 cells treated with TNF- for 2 h (right panels). Here, the individual protein quantity was determined as representative numbers with a text box by using PD Quest 7.3
(Bio-Rad Laboratories) analysis software. Marked circle regions show the proteins that are differentially expressed under the test conditions.

has been investigated by knockdown of protein expression or gene

knockout, the overall levels of all RHO GTPases with which it interacts decreased (Boulter et al., 2010). RHO GTPases play important
roles in cell adhesion (both to other cells and to the matrix), cell
polarity, vesicle trafcking, cell cycle progression, gene expression,

differentiation and aberrant regulation in tumor cells (Sahai and

Marshall, 2002). Furthermore, changes in RHOGDI1 and RHOGDI2
expression levels have been associated with many cancers. However, the changes vary depending on the tumor type (Harding
and Theodorescu, 2010). Thus, down-regulation of RHOGDI1 may

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A. Das et al. / Molecular Immunology 56 (2013) 113122

hamper the cell adhesion, cell polarity, vesicle trafcking, cell cycle
progression, gene expression, and differentiation in TNF--treated
JMJD3-kd THP-1 cells.
We found that NudC was signicantly down-regulated in the
TNF--treated JMJD3-kd THP-1 cells (Fig. 1A). NudC plays an important role in cytokinesis and mitosis in mammalian cells (Aumais
et al., 2003). Depletion of NudC inhibits cell growth and induces
mitotic arrest with multiple mitotic defects, which subsequently
result in cell death through loss of dynein function (Zhou et al.,
2006). Regarding these ndings, NudC may be a potential target for tumor suppression in the TNF--treated JMJD3-kd THP-1
cells. NUDT5, which was down-regulated in TNF--treated JMJD3kd THP-1 cells, has an important role in the hydrolysis of 8-oxo
guanosine diphosphate to 8-oxo guanosine monophosphate and
therefore prevention of misincorporation of 8-oxo guanosine into
RNA. The 8-oxo guanosine containing nucleotides can be incorporated into RNA or DNA, which can cause transcriptional and
replicational errors, respectively (Ishibashi et al., 2005). In addition, recently, Zhang et al. (2012) reported that the cell cycle G1
phase was signicantly delayed and that the cell numbers in both
S and G2/M phases were reduced by NUDT5 suppression through
induction of key proteins that prevent the G1-S transition, including
p53, p16 and Rb. Thus, down-regulation of NUDT5 could have possible roles in inhibiting cell proliferation as well as transcriptional
and replicational errors through incorporation of 8-oxo guanosine
into RNA in TNF--treated JMJD3-kd THP-1 cells. Another signicantly down-regulated protein in TNF--treated JMJD3-kd THP-1
cells is GMFG. GMFG is expressed in a variety of hematopoietic
cells such as T cells, B cells, NK cells, monocytes, and dendritic
cells, as well as hematopoietic progenitor cells including CD34
and CD105 bone marrow cells (Asahara et al., 1997). Ikeda et al.
(2006) demonstrated that, the amino acid sequence of GMFG is
similar to colin, a key regulator of actin cytoskeleton reorganization, which was essential for a large number of cellular processes
such as cytokinesis, endocytosis, and chemotaxis. Inhibition of
actin cytoskeleton reorganization causes impaired cell migration,
which reduces macrophage phagocytosis, and defective lymphocyte development and activation (Bamburg and Wiggan, 2002).
This nding indicates that actin cytoskeleton reorganization is
pivotal for angiogenesis and immune system function. Therefore,
down-regulation of GMFG may have possible roles in inhibiting the
cellular processes and cell migration in TNF--treated JMJD3-kd
THP-1 cells.
Caspase
recruitment
domain
family,
member
14
(CARD)/CARMA2 (CARD14/Bimp3) was down-regulated in TNF-treated JMJD3-kd THP-1 cells (Fig. 1A). Ectopic expression of
CARMA family members induces NF-B activation in most cell lines,
as well as primary cells (Matsumoto et al., 2005). Recently, Scudiero
et al. (2011) reported that CARMA2 is pivotal for the regulation of
NF-kB activation and endoplasmic reticulum (ER) stress-induced
cell death. Regarding these ndings, down-regulation of CARMA2
could be a potential target for the inhibition of NF-B activation
and ER stress-induced cell death in TNF--treated JMJD3-kd
THP-1 cells. Another signicantly down-regulated protein is dUTP
pyrophosphatase in TNF--treated JMJD3-kd THP-1 cells (Fig. 1A).
Induction of dUTP pyrophosphatase causes irreversible DNA
fragmentation and cell death (Ingraham et al., 1986). Thus, control
of intracellular dUTP levels is indispensable for cellular organisms.
Furthermore, there is indirect evidence implicating increases
in intracellular dUTP in the action of cancer chemotherapeutic
agents, including the thymidylate synthase inhibitors and the
folate agonists (e.g., methotrexate and aminopterin). Both agents
promote thymineless cell death through inhibition of thymidylate
synthase and dihydrofolate reductase, which prevents de novo
synthesis of dTTP and causes DNA fragmentation (Curtin et al.,
1991). Thus, down-regulation of dUTP pyrophosphatase may have

possible roles in inhibiting DNA fragmentation and cell death in

TNF--treated JMJD3-kd THP-1 cells. Finally, the results achieved
by the real time RT-PCR and western blot analysis of TRIM5, JMJD3,
and NUDC shown in Fig. 3A and C, illustrated that there was
essentially down-regulation in the expression of above mentioned
mRNA and proteins in TNF--treated JMJD3-kd THP-1 cells than
that of THP1 sc and untreated THP1 sc.
We found that WDR67 protein was signicantly up-regulated
in TNF--treated JMJD3-kd THP-1 cells (Fig. 1B). The WDR67 protein is essential for a variety of cellular functions because of its high
degree of diversity in sequence as well as its multi-domain context.
These functions include signal transduction and transcription regulation, as well as cell cycle control (Li and Roberts, 2001). Here, the
induced expression of WDR67 may be responsible for transcription
regulation of cell cycle control in TNF--treated JMJD3-kd THP-1
cells. Another signicant up-regulated protein was HspB1/Hsp27 in
TNF--treated JMJD3-kd THP-1 cells (Fig. 1B). HspB1 has the ability
to increase the resistance of cells to oxidative injuries (Arrigo, 2001)
and also maintains the mitochondrial membrane potential level
(Paul and Arrigo, 2000). This phenomenon provides stressed cells
with abundant ATP production, which favors the activity of chaperones. HspB1 is also involved in inammation diseases. For example,
these proteins interfere with the TNF- signaling pathway through
their ability to protect against oxidative stress (Mehlen et al., 1995),
and HspB1 also suppresses NF-B activation through an interaction with IKK- and IKK- (Kammanadiminti and Chadee, 2006).
Regarding these ndings, up-regulation of HspB1 could be a potential target for the control of inammatory processes. Up-regulation
would cause inhibition of NF-B- activation and oxidative stress
in TNF--treated JMJD3-kd THP-1 cells where JMJD3-attenuation
could be vital for controlling the HspB1-mediated inammatory
mechanism.
Prohibitin, which has an important role in cellular processes
including the regulation of proliferation and transcription (Wang
et al., 2002), was induced in the TNF--treated JMJD3-kd THP-1
cells (Fig. 1B). Prohibitin sustains anti-oxidant expression (Theiss
et al., 2009a) and has anti-inammatory properties, such as reducing TNF--stimulated NF-kB activation (Theiss et al., 2009b). In
addition, Prohibitin interacts with complex I and subunits of
cytochrome c oxidase of the respiratory chain to regulate their
assembly (Tsutsumi et al., 2009). Thus, up-regulation of prohibitin
in TNF--treated JMJD3-kd THP-1 cells could have a possible
role in anti-inammatory properties through inhibition of NFB-activation where JMJD3-attenuation may prove to be crucial.
Another signicantly up-regulated protein was FBP in TNF- treated JMJD3-kd THP-1 cells (Fig. 1B). FBP has an important role
in most folate transport across cell membranes and high-afnity
binders consisting of the folate receptor (FR), which mediate
folate uptake by endocytosis. Folate is involved in the remethylation of homocysteine to methionine, which is important for the
biosynthesis of S-adenosyl methionine, an essential supplier of
methyl groups for the methylation of many compounds including DNA, RNA, proteins, and phospholipids (Mason and Choi,
2000). Deciency of folate is associated with misincorporation
of uracil instead of thymine into DNA. Misincorporation leads to
increased chromosomal strand breaking (Blount et al., 1997) and
abnormal methylation reactions (Stern et al., 2000). Both pathways are implicated in cancer (Strathdee et al., 2001). Therefore,
up-regulated FBP may have a possible role in inhibiting the chromosomal strand breaking and abnormal methylation reactions in
TNF--treated JMJD3-kd THP-1 cells. Consequently, we validated
expression of selected genes by real time RT-PCR and protein
expression by western blot analysis where WDR67, Prohibitin, and
HspB1 showed up-regulation in TNF--treated JMJD3-kd THP-1
cells than that of THP1 sc and untreated THP1 sc (Fig. 3B and
D).

A. Das et al. / Molecular Immunology 56 (2013) 113122

119

Fig. 2. Effect of molecular and signaling network of proteins for both TNF--treated JMJD3-kd THP-1 cells in comparison with THP1 sc cells. The direct (solid line) and indirect
(broken line) interactions of both up-regulated (red) or down-regulated (white) genes. A direct interaction of both up-regulated or down-regulated genes were associated
with the crucial molecules for inhibition of MAPK- and NF-B-dependent inammatory genes, oxidative stress and transcription, mRNA processing, chromatin remodeling
and nuclear matrix association identied in TNF--treated JMJD3-kd THP-1 cells (A), and networks involved in inammatory disease, respiratory disease and immunological
disease are shown in (B). This dataset was analyzed using Ingenuity Pathway Analysis. The node color is indicative of gene expression, and the color intensity is proportional
to the gene expression fold change. (For interpretation of the references to color in gure legend, the reader is referred to the web version of the article.)

120

A. Das et al. / Molecular Immunology 56 (2013) 113122

Table 3
Ingenuity pathway analyses revealed top six interacting gene networks in JMJD3-kd human leukemia monocyte cells.
No.

Associated network functions

Score

1
2
3
4
5
6

Gene expression, cellular assembly and organization, endocrine system development and function
Inammatory disease, respiratory disease, immunological disease
Gene expression, cellular development, hematological system development and function
Genetic disorder, hematological disease, cellular assembly and organization
Carbohydrate metabolism, small molecule biochemistry, lipid metabolism
Cell death, cellular development, hematological system development and function

29
26
5
3
3
2

IPA analysis was used to identify gene networks, canonical

pathways, and molecular and cellular functions in 2 h TNF-treated JMJD3-kd THP-1 cells. This network analysis can provide
primary information about physical connectivity and functional
relationships between proteins. The functional roles and number
of molecules differentially expressed in TNF--treated JMJD3-kd
THP-1 cells is shown in Table 3. Here, IPA analysis showed that
JMJD3-kd-affected genes are mostly responsible for inammatory
disease, respiratory disease, immunological disease, genetic disorder, and hematological disease in THP-1 cells. The top-scoring
gene networks identied in 2 h TNF--treated JMJD3-kd THP-1
cells were genes involved in inhibition of MAPK- and NF-Bdependent inammatory genes, oxidative stress and transcription,
mRNA processing, chromatin remodeling and nuclear matrix association (Fig. 2A) and networks involved in inammatory disease,
respiratory disease and immunological disease (Fig. 2B). Notably,
the proteomic analysis of 2 h TNF--treated JMJD3-kd THP-1 cells
showed that the up-regulation of tat binding protein, heat shock
protein beta-1, and protein disulde-isomerase A3, along with
the down-regulation of TRIM5, NudC, and other proteins, is signicant for the establishment of the 2 h TNF--treated JMJD3-kd
THP-1 cells. To analyze the pathways related to the inammatory

response, we next checked Interleukin 3 receptor alpha signaling.

IPA and MALDI-TOF-MS analysis revealed that Interleukin 3
receptor alpha was up-regulated in 2 h TNF--treated JMJD3kd THP-1 cells (Table 1). Interleukin 3 receptor alpha activates
receptor-associated Janus tyrosine kinase (JAK1/2) and phosphorylates signal transducer and activator of transcription proteins
(STAT1/3/5/6). Subsequently, STAT1/3/5/6 form heterodimers and
translocate into the nucleus where they bind to specic DNA elements responsible for cell proliferation, cell differentiation and
hematopoiesis. C-jun transcription factor, in combination with cFos, and extracellular signal-regulated kinase (ERK1/2) were also
signicantly affected. Additionally, they are crucial for cell proliferation, cell differentiation and hematopoiesis in 2 h TNF--treated
JMJD3-kd THP-1 cells (Fig. S1).
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molimm.
2013.04.013.
Overall, proteomic MALDI-TOF-MS and IPA analysis uncovered
key proteins and signaling networks potentially responsible for
the suppression of different key inammatory regulators through
JMJD3 attenuation in THP-1 cells. Although we found evidence for
up- and down-regulation of several proteins in MALDI-TOF-MS,

Fig. 3. Validation of selected genes by real-time RT-PCR and western blotting. (A) TRIM5, JMJD3 and NUDC gene expression was signicantly down-regulated in TNF--treated
JMJD3-kd THP-1 cells and (B) up-regulated gene expression was WDR67, Prohibitin and HspB1 after 2 h TNF treatment in JMJD3-kd THP-1 cells. *P < 0.05 value compared
with THP-1-sc cells. Values represent mean SD (N = 3). (C) WB analysis revealed suppression of JMJD3, TRIM5 and NUDC protein expression in 2 h TNF- treatment JMJD3-kd
THP-1 cells. (D) HspB1 and FBP protein expression were up-regulated after 2 h TNF- treatment JMJD3-kd THP-1 cells. -Actin expression was detected as a control. Data
shown are representative of three independent experiments.

A. Das et al. / Molecular Immunology 56 (2013) 113122

extensive in vivo experimentation with JMJD3 mutant mice can

conrm this hypothesis. At the same time, we could not exclude
the possibility of the JMJD3 close paralog ubiquitously transcribed
tetratricopeptide repeat X chromosome (UTX) being involved in
regulation of other proteins in THP-1 cells. Further studies are
needed to conrm the UTX involvement in the regulation of inammatory mechanisms.
4. Conclusion
The results presented here in suggest that a wide spectrum of
cellular responses is induced in TNF--treated JMJD3-kd THP-1
cells. JMJD3-kd THP-1 cells used in this study showed downregulation of some proteins after 2 h of TNF- treatment, especially
TRIM5, GPx, RHOGDI1, NudC, NUDT5, GMFG, CARMA2, and dUTP
pyrophosphatase. This down-regulation could inhibit NF-B and
other inammatory cellular processes that are important in cancer
and other human diseases. In addition, the signicant up-regulation
of WDR67, HspB1, prohibitin, and FBP may have important antiinammatory properties through inhibition of NF-B-activation,
oxidative stress and chromosomal strand breaking. Moreover, IPA
analysis identied interacting proteins that were differentially
expressed in TNF--treated JMJD3-kd THP-1 cells involved in
inammatory disease, respiratory disease, immunological disease,
genetic disorder and hematological disease. In summary, we propose that these proteomic proles have identied some important
proteins that could signicantly regulate inammation in TNF-exposed JMJD3-kd THP-1 cells.
Conict of interests
The authors declare that they have no conicts of interest.
Acknowledgements
This work was supported by the National Research Foundation
of Korea Grant funded by the Korean Government (No. 20120009212).
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