BioChip J.

(2013) 7(1): 75-84

DOI 10.1007/s13206-013-7112-0

Original Research

Identification of survival factors in LPS-stimulated anthrax

lethal toxin tolerant RAW 264.7 cells through proteomic
approach
Amitabh Das1,, Nando Dulal Das1,, Ji Hyun Park1, Hyung Tae Lee1, Mi Ran Choi1,
Kyoung Hwa Jung1 & Young Gyu Chai1
Received: 16 August 2012 / Accepted: 12 October 2012 / Published online: 20 March 2013
The Korean BioChip Society and Springer 2013

Abstract Anthrax lethal toxin (LeTx; a combination

of protective antigen and lethal factor) is secreted by
the vegetative cells of Bacillus anthracis and is cytotoxic for certain macrophage cell lines. First-time exposure of murine macrophage cells (RAW 264.7) to lethal
toxin (LeTx) (0.1+0.1 mg/mL) caused extensive cell
death with a survival rate of approximately 40%, but
upon secondary exposure to LeTx and lipopolysaccharide (LPS) (1 g/mL), after a few passages, these
cells had a survival rate of approximately 100%. The
present study assessed protein expression changes after
LPS exposure to LeTx-intoxication-resistant RAW
264.7 cells. To analyze the protein expression profile
of LPS-treated LeTx-intoxication-resistant RAW 264.7
cells, we employed matrix-assisted laser desorption/
ionization-time-of-flight mass spectrometry (MALDITOF MS), and later, Ingenuity Pathways Analysis
(IPA) was applied to establish the molecular networks.
Among the differentially expressed proteins were
voltage dependent anion channel 1, jip3 protein, heat
shock protein 4, tubulin beta, 26S protease regulatory
subunit 4, and DNA polymerase delta subunit 4 (DNA
pol) were significant. The molecular network signatures and functional proteomics obtained in this study
may facilitate the evaluation of potential pathways
such as PI3K signaling, NF-B signaling, and LPSinduced MAPK signaling for the recovery of LPS1

Division of Molecular & Life Science, Hanyang University,

Ansan 426-791, Korea

These authors contributed equally to this work.

Correspondence and requests for materials should be addressed to
Y.G. Chai ( ygchai@hanyang.ac.kr)

induced LeTx-intoxication-resistant RAW 264.7 cells.

Keywords: Anthrax lethal toxin (LeTx), Cell survival,
Ingenuity Pathway Analysis (IPA), Lipopolysaccharides
(LPS), LeTx-intoxication-resistant RAW 264.7 cell,
MALDI-TOF MS

Introduction
Anthrax lethal toxin (LeTx) is a binary toxin of Bacillus
anthracis composed of protective antigen and lethal
factor. Protective antigen (P) is a molecular transporter
promoting the receptor-mediated entry and release of
lethal factor (Lx) into the cytosol. Lx is a zinc metalloprotease that cleaves the N-terminal end of mitogenactivated protein kinase (MAPK) kinases (MEK) 1 to
7, with the exception of MEK5, resulting in the inactivation of most of their downstream signaling cascades.
P binds to one of two surface receptors: anthrax receptor 1 (also known as the tumor endothelial marker 8)
and anthrax receptor 2 (also known as the capillary
morphogenesis gene-2). While anthrax receptor 2 is
widely distributed in human tissues, anthrax receptor
1 is expressed abundantly in macrophages and is also
found in other cells, including endothelial cells and
several tumor cells1,2. It has been reported that NTP
ase domain (NACHT)-leucine-rich repeat and pyrindomain-containing protein 1b (NALP1b) in mice acts as
the host factor that confers rapid LeTx cytotoxicity3.
Moreover, human macrophages lacking NALP1b are
resistant to rapid necrotic cell death induced by LeTx.
Instead, LeTx was shown to cause delayed apoptotic

76

cell death of differentiated macrophages and to inhibit cell proliferation and differentiation, most likely
mediated through MAPK inhibition4. However, the
exact mechanism of anthrax pathogenesis by the inhibition of cell proliferation by LeTx has yet to be elucidated. Interestingly, recovery from prolonged MEKcleaving Lx activity required cell proliferation, which
was mediated in some cells through an adaptive response by the induction of the phosphatidylinositol 3kinase (PI3K)/Akt/GSK3 signaling pathway. This suggests that the recovery from cellular LeTx toxicity
somehow depends on the activation of PI3K/Akt pathway5. Although an impaired immune response, cell
lysis due to the loss of ions and the degradation of survival factors are critical components of LeTx-induced
cell death6 in macrophages, a short exposure to LeTx
primarily down-regulates NF-B and GSK3-regulated
genes7. Several kinases, including PI3K/Akt kinase,
signal through NF-B to promote cell survival. In
non-stimulated cells, the basal nuclear NF-B levels
may regulate the expression of certain genes required
for cell survival8.
The present study examines the possibility that the
comparative differential induction on the protein expression profile of 2 h LPS-treated LeTx-intoxication-resistant macrophages cells using two-dimensional polyacrylamide gel electrophoresis (2D)/matrix-assisted
laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) that have developed resistance to LeTx-intoxication involving the induction of
possible survival factors. In addition, the signaling
pathways and molecular networks induced by 2 h LPS
treatment of LeTx-resistant RAW 264.7 cells were
determined by IPA, to define those targets associated
with resistance to LeTx intoxication. Notably, the
molecular network characterized by IPA analysis
showed that LPS-treated LeTx-intoxication-resistant
RAW 264.7 cells induced the protein expression of
tubulin beta, heat shock protein 4 (hsp 4), voltage
dependent anion channel 1 (VDAC1) for the involvement of CREB1 (CAMP responsive element binding
protein 1), activating transcription factor 2 (ATF2),
NF-B and JNK (c-Jun N-terminal kinases), which
are essential for the recovery of murine macrophage
cells. The differentially expressed proteins included
VDAC1, jip3 protein, hsp 4, tubulin beta, Impa1, 26S
protease regulatory subunit 4, calcium-binding 140K
protein, DNA polymerase delta subunit 4, tripeptidyl
peptidase I, protein disulfide-isomerase (PDI), macrophage-capping protein (CAPG), carbonic anhydrase 6
(Car6), and caspase activation inhibitor. Overall, LPSinduced LeTx-intoxication-resistant cells showed a
pattern of differentially expressed proteins which suggest that resistance may be mediated via a number of

BioChip J. (2013) 7(1): 75-84

mechanisms: induced cell proliferation, anti-apoptosis,

activation of survival signaling pathways, cell cycle
progression, differentiation, and prevention of caspase
activation. This study highlights the possible proteomic based markers that have important roles in cell
survival in the LPS-treated LeTx-intoxication-resistant
cells.

Results and Discussion

LeTx-intoxication-resistant cells and RAW 264.7 cells
were treated with LPS for 2 h and their protein expression patterns were analyzed using a 2DE (pH 3.0-10)
technique. The first dimension IEF separation was performed on an IPG strip and subsequently, the second
dimension was performed on 10-16% SDS-PAGE. We
used alkaline silver staining that detected approximately 1800200 spots per gel. Gel images were analyzed using PDQuest 7.3 image software, which permitted a comparison of the expression patterns of proteins after LPS 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 43 up and down-regulated proteins.
The 2 h LPS exposure resulted in the significant upregulation of the following proteins in LeTx-intoxication-resistant RAW 264.7 compared with normal RAW
264.7 cells: VDAC1, jip3 protein, hsp 4, tubulin beta
( tubulin), 26S protease regulatory subunit 4, DNA
polymerase delta subunit 4, and tripeptidyl peptidase
I (TPPI) (Table 1). In addition, a few down-regulated
proteins were detected, with PDI, CAPG, car6, and
caspase activation inhibitor, all of which showed significant differences (Table 2).
VDAC1, which has an important role in cytochrome
c-mediated cell survival13, was induced in the LPStreated intoxication-resistant cells (Figure 1A). The
VDAC1 N-terminal region interacts with hexokinase
and Bcl2 in a manner that confers protection against
apoptosis14. Thus, up-regulation of VDAC1 could
contribute the cytochrome c mediated cell survival in
the LPS-treated LeTx-intoxication-resistant cells. Another protein significantly up-regulated in the LeTxintoxication-resistant cells is JIP3 (Figure 1A). The JIP
group of scaffold proteins (also known as IB/JSAP)
bind to c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase 7 (MKK7), and to members of
the mixed-lineage protein kinase (MLK) group that
activate the JNK15, which is involved in cell proliferation16. Although it is established that JNK contributes
to some apoptotic responses, it is not clear that apoptosis represents the only functional consequence of JNK

BioChip J. (2013) 7(1): 75-84

77

Table 1. Up-regulated proteins in 2 h LPS-treated LeTx-intoxication-resistant RAW 264.7 cells.

RAW 264.7 cells

Ratio

Estimated Z

Access
number

44624
15085
14126
9796
8412
3798
3769
341

44624
15085
14126
9796
8412
3798
3769
341

0.93
2.29
2.38
1.09
0.89
0.4
1.82
0.99

gi23503505
gi7106439
gi5051881
gi313482801
gi148674154
gi148701657
gi6679501
gi148706216

158

9538

60.36

0.93

gi21312410

5602
6307
4303
7501
6210
5501
5401
4701
4202
3307
6212
4304

119
248
118
433
621
2983
2785
950
570
474
740
2371

5175
10081
2147
5270
4428
21183
16993
4518
2207
1659
2577
7825

43.48
40.64
18.19
12.17
7.13
7.1
6.1
4.75
3.87
3.5
3.48
3.3

2.03
1.14
0.37
2.43
0.48
2.43
0.92
2.43
1.15
2.43
1.33
0.78

gi148684872
gi296061338
gi148692681
gi148682452
gi124249212
gi2078001
gi26006117
gi1083243
gi21312396
gi29145083
gi34147238
gi148671242

5304
4307
6301
6502

751
2302
9230
13047

2406
4835
12923
15657

3.2
2.1
1.4
1.2

1.37
1.49
0.66
1.33

gi14198211
gi148701664
gi168984324
gi148706214

Protein name

Spot No.

UDP-glucuronyltransferase-S
Tubulin beta-5 chain
JIP-3
Hypothetical protein LOC100040022
hnRNP-associated with lethal yellow, isoform CRA_g
Heat shock protein 4, isoform CRA_d
26S protease regulatory subunit 4
Ubiquitin-like, containing PHD and RING finger
domains, 1, isoform CRA_c
Tumor necrosis factor alpha-induced protein 8-like
protein 2
Tripeptidyl peptidase I, isoform CRA_b
Immunoglobulin heavy chain variable
mCG18674, isoform CRA_b
mCG7898, isoform CRA_b
Hypothetical protein LOC72277
Vimentin
mKIAA0219 protein
Calcium binding 140k protein - mouse (fragment)
DNA polymerase delta subunit 4
Impa1 protein
Downregulated in renal cell carcinoma
Proteasome (prosome, macropain) 26S subunit,
ATPase 2, isoform CRA_b
Tnfaip2 protein
Voltage-dependent anion channel 1, isoform CRA_a
Enolase 1, alpha non-neuron
Ubiquitin-like, containing PHD and
RING finger domains, 1, isoform CRA_a

6401
6302
3506
7401
6308
4601
5404
3306

1
1
1
1
1
1
1
1

6302

NC

PLxL

Table 2. Down-regulated proteins in 2 h LPS-treated LeTx-intoxication-resistant RAW 264.7 cells.

Protein name

Spot No.

Apoptosis, caspase activation inhibitor, isoform CRA_b

Protein disulfide-isomerase A3 precursor
DNA segment, Chr 14, ERATO Doi 500, expressed
Protein midA homolog, mitochondrial precursor
mCG1047313
CAPG_MOUSE (Macrophage-capping protein)
Prolactin-7D1 precursor
Car6 protein
RIKEN cDNA 9430070O13 gene

3202
3509
4310
3508
3309
3308
3404
5408
4501

RAW 264.7 cells

NC

PLxL

40377
7680
5573
4313
1068
738
2576
4274
2736

1
1
4.8
1
1
1
480
268
116

Ratio

Est Z

Access
number

0
0
0
0
0
0
0.18
0.06
0.042

2.43
2.43
2.43
2.43
1.94
2.43
2.43
2.43
2.43

gi148695899
gi112293264
gi148704395
gi158937256
gi148707568
gi729023
gi6755102
gi116283330
gi223462287

NC indicates untreated control; Estd Z, estimated Z score as a measure of confidence for Profound-based identification

activation. This is obvious when one considers that

most stimuli that activate JNK do not cause apoptosis.
For example, most forms of environmental stress do
not cause apoptosis under conditions that are sufficient
for JNK activation. This is partly because the JNKdependent apoptotic signaling pathway can be blocked
by the activation of survival signaling pathways17.
Examples of these survival pathways include NF-B,

Akt/PKB, and ERK. We have reported that LeTx-intoxication-resistant cells showed abundant expression of
different NF-B genes, especially p100, p50 and p659.
Cells may interpret transient JNK activation as a survival signal because of the activation state of other signaling pathways within the intoxication-resistant cells.
In addition, it has been reported that integrin-mediated
survival signaling can be transduced by the JNK path-

78

BioChip J. (2013) 7(1): 75-84

PLxL

RAW 264.7

PLxL

RAW 264.7

TPPI

DNA pol

JIP-3

VDAC-1

(A)

PLxL (LPS-treated LeTx-intoxication-resistant cells)

PLxL

RAW 264.7

PLxL

RAW 264.7

CAPG
Caspase activation inhibitor

Car 6

PDI

(B)

PLxL (LPS-treated LeTx-intoxication-resistant cells)

Figure 1. Enhanced segments of the two-dimensional gel electrophoresis gel derived from different treated samples: (A and B)
LPS-treated LeTx-intoxication-resistant RAW 264.7 cells (left panels) and RAW 264.7 cells (right panels). Marked regions show
the proteins that are differentially expressed under the test conditions.

BioChip J. (2013) 7(1): 75-84

way18. These observations are consistent with the hypothesis that JNK may mediate survival signaling
under specific circumstances. Given that we have
already shown the induced expression of various NFB genes in LeTx-intoxication-resistant cells, the upregulated JIP3 in these cells may scaffold with other
proteins such as JNK, NF-B, and ERK that may
facilitate cell survival. Expression of PHD and RING
finger domains was increased in after LPS exposure
in LeTx-intoxication-resistant RAW cells. The PHD
domain containing protein Np95 is a cell cycle-regulated and histone-binding protein expressed only in
proliferating cells 19 and over-expression of Np95ICBP90 induces decondensation of chromocenters20.
In this case, up-regulation of PHD domains could
promote the decondensation of chromocenters or cell
proliferation in LPS-treated LeTx-intoxication-resistant RAW cells.
We found that hsp4 protein (70 kDa) was significantly up-regulated in the LPS-treated LeTx-intoxicationresistant RAW 264.7 cells. Some studies have shown
that over-expression of hsp27, hsp70, hsp60 or hsp90
inhibited apoptosis and prevented caspase activation
in many different cellular models reacting to a variety
of cellular stresses, including accumulation of misfolded proteins, reactive oxygen species (ROS) or DNA
damage21. In addition, Wang et al.22 reported that hsp
110, hsp70 and hsp25 formed a large complex and
directly interacted with one another in promoting cell
proliferation, cell cycle and protection from environmental stress. Thus, over expression of hsp4 could
contribute a possible role in prevention of caspase
activation and apoptosis as well as the induction of
cell proliferation after 2 h of LPS-treated LeTx-intoxication-resistant RAW 264.7 cells. Another significantly up-regulated protein was tubulin. This protein,
which is a major component of microtubules (MTs),
is essential for a variety of cellular functions, including the maintenance of cell shape and cell polarity,
intracellular transport, cell mitosis, and meiosis. MT
networks are intrinsically highly dynamic and undergo dramatic reorganization during the cell cycle23.
Here, the induced expression of tubulin in 2 h LPStreated LeTx-intoxication-resistant cells could be
responsible for cellular reorganization during the cell
cycle. When cells enter mitosis, the interphase MT
network is rapidly disassembled and reorganized into
the mitotic spindle. The precise regulation of microtubule assembly and disassembly at both kinetochores
and centrosomes is thought to be important for the
maintenance of spindle structure and chromosome
segregation during mitosis24. IMPA 1, which was upregulated in LPS-treated LeTx-intoxication-resistant
RAW 264.7 cells, has an important role in the phos-

79

phatidylinositol signaling system, catalyzing the dephosphorylation of various myoinositol monophosphates to free myo-inositol25. Myo-Inositol plays an
important role as the structural basis for a number of
secondary messengers including inositol phosphates,
phosphatidylinositol (PI) and phosphatidylinositol
phosphate (PIP) lipids in eukaryotic cells which have
crucial role in diverse cellular functions, such as cell
growth, cell migration, endocytosis, cell differentiation,
cell signaling, membrane trafficking and transcription
mediated gene expression26,27. So, up-regulated IMPA
1 after 2 h LPS exposure in LeTx-intoxication-resistant
RAW cells could have possible roles in cell proliferation, migration as well as cell differentiation.
The 26 S proteasome is largely responsible for the
ATP and ubiquitin dependent degradation of misfolded
and other short-lived proteins28 and the misfolded or
other short-lived proteins were could injure or kill the
cell29. Thus the over expression of 26 S proteasome
after 2 h LPS exposure in LeTx-intoxication-resistant
RAW cells could protect the cell through degradation
of misfolded or other short-lived proteins. Another
up-regulated molecule was the calcium-binding 140 K
protein. Calcium binding proteins participate in calcium cell signaling pathways by binding to Ca2+. The
most ubiquitous Ca2+-sensing protein, found in all
eukaryotic organisms including yeasts, is calmodulin.
Intracellular storage and release of Ca2+ from the sarcoplasmic reticulum is associated with the high-capacity, low-affinity calcium-binding protein calsequestrin.
With their role in signal transduction, calcium-binding proteins contribute to all aspects of the cells functioning, from homeostasis to learning and memory.
So, up-regulated Calcium-binding proteins after 2 h
LPS exposure in LeTx-intoxication-resistant RAW
cells may regulate cell functional properties as well
as cell homeostasis. Another up-regulated protein was
the DNA polymerase delta subunit (pol) (Figure 1A).
The B family of DNA polymerases (pol), which comprises pol, pol, and pol, is essential for the replication of the eukaryotic genome. The mammalian pol
is composed of at least four subunits30. Two subunits
form a tightly associated core heterodimer consisting
of the catalytic p125 subunit, which has both 5 to 3
polymerase and 3 to 5 exonuclease activities, and
the p50 subunit. The enzymatic activity of polis upregulated when quiescent cells are induced to proliferate31 and p125 protein levels show a 2-3-fold increase
at the G1/S phase of the cell cycle32. Additionally, p125
subunit interacts with and becomes phosphorylated
by cyclin D3/Cdk4 and cyclin E/Cdk233, thus suggesting a possible role in S phase checkpoint control. Upregulated pol in 2 h LPS-treated LeTx-intoxicationresistant RAW 264.7 cells could involve in cyclin D3

80

and cyclin E-dependent progression of cell cycle. Another up-regulated protein was TPPI (Figure 1A) in
LPS-treated LeTx-intoxication-resistant RAW 264.7
cells. TPPI is an aminopeptidase that removes tripeptides from the unmodified N-terminus of small polypeptides34. TPPI is synthesized in the endoplasmic
reticulum (ER) as a preproenzyme with a 19-amino
acid (aa) residue signal peptide35. Upon its translocation into the ER lumen, the signal peptide is removed,
and the resulting 544-aaproenzyme is N-glycosylated
and subjected to assistedproper protein folding and
cell-cell interactions36. So, up-regulated TPPI after 2
h LPS exposure in LeTx-intoxication-resistant RAW
cells may regulate the cellular environment by enhancing protein folding or cell-cell interactions.
PDI was significantly down-regulated in LPS-treated
LeTx-intoxication-resistant-RAW 264.7 cells (Figure
1B). PDI is a multi-domain, multi-functional member
of the thioredoxin superfamily that catalyzes thioldisulfide oxidation, reduction and isomerization, the
last of which occurs directly through intramolecular
disulfide rearrangement or through cycles of reductionand oxidation37. PDI behaves as a chaperone, inhibiting the aggregation of misfolded proteins. However, PDI displays unusual behavior for a folding assistant. At concentrations of 1/5-1/10 that of its substrate,
PDI precipitates several normally soluble, misfolded
proteins by inducing aggregation38. The ability of PDI
to specifically facilitate substrate aggregation, has
been termed anti-chaperone behavior39. At high concentrations of PDI, the intermolecular cross-linking
interactions needed to form insoluble complexes are
out-competed by excess PDI38. Thus down-regulation
of PDI in 2 h LPS-treated LeTx-intoxication-resistant
RAW 264.7 cells may involve inhibiting the aggregation of misfolded proteins. Another down-regulated
protein is CAPG (Figure 1B). CAPG is a member of
the gelsolin/villin family of actin-regulatory proteins
that in humans is encoded by the CAPG gene. This
protein reversibly blocks the barbed ends of F-actin
filaments in a Ca2+ and phosphoinositide-regulated
manner40. In addition, Sun et al.41 demonstrated that
CAPG and related proteins are poised to coordinated
membrane signaling with actin filament dynamics following cell stimulation thus suggesting that the suppressed expression of CAPG in 2 h LPS-treated LeTxintoxication-resistant cells may have possible roles in
cell signaling as well as in membrane trafficking.
Car 6 was also down-regulated in LPS-treated LeTxintoxication-resistant cells RAW 264.7 cells (Figure
1B). It is a carbonic anhydrase that in humans is encoded by the CA6 gene. Sun et al.41 reported that CA6 is
a target gene for a stress-inducible transcription factor (CHOP), and they speculate on the possible physio-

BioChip J. (2013) 7(1): 75-84

Table 3. Molecular and Cellular Functions in 2 h LPS-treated

LeTx-intoxication-resistant RAW 264.7 cells.
Top functions

No. of
molecules

p-value

Cell Survival and Death

Cell-To-Cell Signaling
and Interaction
Cellular Assembly and
Organization
Cellular Compromise
Cellular Function and
Maintenance

11
8

1.75E-04-4.08E-02
5.21E-04-4.95E-02

1.28E-03-2.41E-02

5
5

1.28E-03-3.66E-02
1.28E-03-4.76E-02

logical role that an intracellular form of carbonic anhydrase may play in cellular adaptation to stress. So,
down-regulated expression of carbonic anhydrase 6
may protect the cells by suppression of cellular stress
in 2 h LPS-treated LeTx-intoxication-resistant cells.
Caspase activation inhibitor was another protein significantly down-regulated in LPS-treated LeTx-intoxication-resistant RAW 264.7 cells (Figure 1B). Caspases
are an evolutionarily conserved family of aspartatespecific cysteine proteases with central functions in
the apoptotic and inflammatory signaling pathways42.
However, non-apoptotic functions of caspases involve
both proteolysis exerted by their catalytic domains
and non proteolytic functions exerted by their prodomains. The regulation of these non-proteolytic functions by the caspase-like decoy molecules c-FLIP (cellular caspase-8 (FLICE)-like inhibitory protein), human
caspase-12, COP, INCA and ICEBERG provides support for the involvement of caspases in the regulation
of cell survival, proliferation, and differentiation43.
Thus the down regulation of caspase activation inhibitor could affect cell survival, proliferation, and differentiation in 2 h LPS-treated LeTx-intoxication-resistant RAW 264.7 cells.
IPA analysis was used to identify gene networks,
canonical pathways, and molecular and cellular functions in 2 h LPS-treated LeTx-intoxication-resistant
RAW 264.7 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 LPS-treated LeTx-intoxication-resistant
RAW 264.7 cells is shown in (Table 3). The top-scoring
gene networks identified in 2 h LPS-treated LeTx-intoxication-resistant RAW 264.7 cells were genes involved
in cell survival, death, cell-to-cell signaling, interaction,
organization, and maintenance. The genes belonging
to the top scoring networks are involved in LPS-stimulated MAPK signaling, PI3/AKT signaling, ERK/
MAPK signaling, and NF-B signaling pathways (displayed in Figure 2) in 2 h LPS-treated LeTx-intoxica-

BioChip J. (2013) 7(1): 75-84

81

Figure 2. Molecular and signaling network of proteins for LPS-treated LeTx-intoxication-resistant RAW 264.7 cells in comparison
with RAW 264.7 cells. The direct (solid line) and indirect (broken line) interaction of both up-regulated (red) or down-regulated
(green) genes where mostly up-regulated genes were associated with LPS-stimulated MAPK signaling,PI3/AKT signaling, ERK/
MAPK signaling, and NF-B signaling identified in LPS-treated LeTx-intoxication-resistant RAW 264.7 cells. 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. ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PI3,
Phosphoinositide 3-kinase.

tion-resistant RAW 264.7 cells. Notably, the proteomic

analysis of 2 h LPS-treated LeTx-intoxication-resistant RAW 264.7 cells showed the up-regulation of
tubulin beta, hsp 4, VDAC1. This up-regulation of
tubulin beta, hsp 4, VDAC1 with other proteins is
significant for the establishment of the LPS-stimulated
MAPK signaling, PI3/AKT signaling, ERK/MAPK
signaling pathways, in which the involvement of
CREB1, activating transcription factor 2 (ATF2), NFB and JNK is essential (Figure 2).
Overall, proteomic MALDI-TOF-MS and IPA analysis uncovered key proteins and signaling networks
that may be responsible for the establishment of LPSinduced LeTx-intoxication resistance of macrophages.
Although we found evidence for up and down regulation of several proteins in MALDI-TOF-MS, extensive
research is needed to determine the exact molecular

mechanisms of proteins in LeTx-intoxication-resistant

macrophages that have important roles in cell survival.

Conclusions
The results presented herein suggest that a wide spectrum of cellular responses is induced in LPS-treated
LeTx-intoxication-resistant RAW 264.7 cells. LeTxintoxication-resistant RAW 264.7 cells used in this
study were a resistant population of normal RAW 264.7
cells in which some proteins, especially VDAC1, tubulin
beta, JIP-3, hsp4, 26S protease regulatory subunit 4,
tripeptidyl peptidase I, and DNA polymerase delta
subunit 4, were abundantly expressed after 2 h of LPS
exposure that could activate survival-promoting signaling. In addition, the significant down-regulation of

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PDI, CAPG, car6, and caspase activation inhibitor

may have important roles in chaperone behavior, membrane signaling pathway, cell survival, proliferation
and differentiation. Moreover, IPA analysis identified
interacting proteins that were differentially expressed
in LPS-treated LeTx-intoxication-resistant RAW 264.7
cells involved in LPS-stimulated MAPK signaling,
PI3/AKT signaling, ERK/MAPK signaling as well as
NF-B signaling pathways. Taken together, we propose that these proteomic profiles have identified some
of the important proteins that could contribute to recovery from LeTx-intoxication in resistant RAW 264.7
cells with LPS exposure.

Materials and Methods

Reagents

Bacillus anthracis protective antigen (P), lethal factor

(Lx), and lipopolysaccharides (LPS) were from Sigma-Aldrich (USA). RPMI 1640, fetal bovine serum
(FBS), and penicillin-streptomycin (PS) were purchased from Gibco Life Technologies (UK).
Establishment of LeTx-intoxication-resistant RAW
264.7 cells and treatments

Establishment of LeTx-intoxication-resistant RAW

264.7 cells was performed as described in our previous
paper9. The murine macrophage cell line RAW 264.7
was originally obtained from the American Type Culture Collection (Manassas, VA). Cell cultures were
maintained at sub-confluence at 37
C in a 5% CO2
humidified atmosphere using RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 100
IU/mL penicillin and 10 g/mL streptomycin. One
day prior to treatment, cells were seeded at 4106
cells/100-mm plate (NuncTM, Kamstrupvej, Denmark).
Both RAW 264.7- and LeTx-intoxication-resistant
RAW 264.7 cells were treated with LPS (1 g/mL) for
aperiod of 2 h under normal culture conditions.

BioChip J. (2013) 7(1): 75-84

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 methods10. 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. The relative spot
volume of each protein, compared with 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 previously described by11, using modified porcine
trypsin (Promega, Madison, WI).
Matrix-assisted laser desorption/ionization-time-offlight mass spectrometry and database analysis

Peptide analysis was performed using an Ultraflex

matrix-assisted laser desorption/ionization (MALDI)time-of-flight (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 20kV injection pulse for duration of flight 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
identification employing a Bayesian algorithm based
on peptide mass fingerprinting data. The presence of
signal peptides was considered when such information
was available in the corresponding National Center
for Biotechnology Information GenPept format flatfile (www.ncbi.nlm.nih.gov/Entrez/batch.html). The
following input parameters: mass tolerance-1, iodoacetamide modifications for amino acid residues, were
applied. The spectra were internally calibrated using
trypsin autodigestion ion peaks (m/z 842.510 and
2211.1046).

Protein sample preparation

Proteins were extracted in lysis buffer (9.5 M urea,

2.5% 3-[(3-cholamidopropyl) dimethylammonio]-1propane 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 described10.
The protein concentrations of ten-fold dilutions of the
samples were measured using the Bradford assay (Sigma-Aldrich Inc., USA), with BSA (2 mg/mL; Pierce
Biotechnology, Inc., Rockford, IL) as a standard.

Canonical pathway analysis of datasets

IPA (Ingenuity Systems [www.ingenuity.com], Mountain View, CA, USA) was conducted to determine the
most significant canonical pathways in the datasets.
Detailed methods for this procedure have been described previously12. In brief, genes from the dataset
were considered for literature review. The significance
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 num-

BioChip J. (2013) 7(1): 75-84

ber 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 identifier 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.
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 from the 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).
Acknowledgements This study was supported by Science and Technology Research Grant number HY-2010N from Hanyang University, Korea.
Conflict of interests The authors declare that they have
no conflict of interests.

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