Open Journal of Cell Biology, 2012, 2, 21-31
doi:10.4236/ojcb.2012.22002 Published Online December 2012 (http://www.SciRP.org/journal/ojcb)
Helicobacter pylori Induction in Gastric Mucosal
Prostaglandin and Nitric Oxide Generation Is Dependent
on MAPK/ERK-Mediated Activation of IKK-β
and cPLA2: Modulatory Effect of Ghrelin
Bronislaw L. Slomiany, Amalia Slomiany
Research Center, University of Medicine and Dentistry of New Jersey, Newark, USA
Received July 17, 2012; revised August 25, 2012; accepted September 9, 2012
ABSTRACT
Among the key factors defining the extent of gastric mucosal inflammatory involvement in response to H. pylori is the
excessive generation of prostaglandin (PGE2) and nitric oxide (NO), caused by the overexpression of cyclooxygenase-2
(COX-2) and inducible nitric oxide synthase (iNOS), and triggered by the activation of MAPK/JNK, p38 and ERK, and
nuclear translocation of the cognate transcription factors. In this study, we report on the role of MAPK/ERK in the
regulation of H. pylori LPS-induced gastric mucosal expression of COX-2 and iNOS. We show that ERK activation by
the LPS leads to phosphorylation of the inhibitory κB kinase-β (IKK-β) and cytosolic phospholipase A2 (cPLA2), and is
reflected in the upsurge in NF-κB nuclear translocation, induction in COX-2 and iNOS expression, and up-regulation in
cPLA2 activity. The modulatory effect of peptide hormone, ghrelin, on the LPS-induced changes, although associated
with further enhancement in ERK, IKK-β and cPLA2 phosphorylation, was reflected in the suppression of IKK-β and
cPLA2 activity through S-nitrosylation. While the effect of ghrelin on S-nitrosylation was susceptible to suppression by
the inhibitors of Src/Akt pathway, the inhibition of ERK activation caused the blockage in IKK-β and cPLA2 phos-
phorylation as well as S-nitrosylation. Taken together, our data show that H. pylori-induced ERK activation plays a
critical role in up-regulation of gastric mucosal PGE2 and NO generation at the level of IKK-β and cPLA2 activation,
and that ghrelin counters these proinflammatory consequences of the LPS through Src/Akt-dependent S-nitrosylation.
Keywords: H. pylori; Gastric Mucosa; Ghrelin; PGE2; NO; COX-2; iNOS; ERK; IKKβ; cPLA2; S-Nitrosylation
1. Introduction
Infection with Helicobacter pylori is well-establish risk
factor in etiology of gastric disease, and the excessive
generation of prostaglandin (PGE2) and nitric oxide (NO)
caused by the overexpression of cyclooxygenase-2 (COX-2)
and inducible nitric oxide synthase (iNOS) are consid-
ered of primary importance in defining the extent of gas-
tric mucosal inflammatory involvement [1-6]. The sig-
naling events underlying the up-regulation in PGE2 and
NO generation indicate that H. pylori cell wall lipopoly-
saccharide (LPS), like that of other Gram-negative bacteria,
is capable of triggering the stimulation of Toll-like re-
ceptor-4 (TLR-4), which then through a series of down-
stream effectors causes the activation of transcriptional
factors that exert control over iNOS and COX-2 gene
expression [2,7-9].
While the induction of iNOS gene by LPS has been
convincingly linked to transcriptional factor NF-κB acti-
vation [9-11], the nature of factors involved in transcrip-
tional regulation of COX-2 expression is less apparent
[7,12-14]. Depending on the cell type, at least four cen-
tral response elements have been implicated in the regu-
lation of COX-2 expression. These include, transcrip-
tional factor, NF-κB, activator protein-1(AP-1), cAMP
response element-biding protein (CREB), and CCATT/
enhancer-binding proteins (C/EBP) β and δ [7,13-17].
Moreover, LPS is a potent activator of mitogen-activated
protein kinase (MAPK) cascade, including extracellular
signal regulated kinase (ERK), c-Jun N-terminal kinase
(JNK) and p38 kinase, which in turn exert their control
over transcription factors activation through phosphory-
lation [12,14-16]. Indeed, we have shown recently that
stimulation of gastric mucosal cells with H. pylori LPS
elicits the activation of all three MAPK subtypes (JNK,
p38, and ERK), and have linked the involvement of
JNK/p38 in the transcription factor AP-1 activation [17].
Studies into the role of LPS-elicited ERK activation in
the regulation of factors linked to the induction of COX-2
and iNOS expression indicate the involvement of MAPK/

B. L. SLOMIANY, A. SLOMIANY 22
ERK in the activation of transcriptional factors, C/EBP
and CREB, implicated in the control of COX-2 expres-
sion, as well as the processes associated with the en-
hancement in NF-κB nuclear translocation and the induc-
tion of iNOS expression [14,15,18-20]. Moreover, H.
pylori LPS-induced ERK activation plays an essential
role in the phosphorylation cPLA2, that facilitates the
enzyme translocation form cytosol to membrane to gain
access to phospholipid substrates for the increase in AA
release [21]. The literature data, furthermore, point to
similarities in the transcriptional regulation of COX-2
and cPLA2 expression; the gene locus of cPLA2 is located
on chromosome I in the proximity of COX-2 gene, and
the increased level of AA affects the COX-2 expression
[22-24].
A substantial body of information also suggests that the
systems involved in transcription factors activation are
regulated through S-nitrosylation [9,25-27]. Indeed, S-
nitrosylation has been linked to the processes of COX-2
and cPLA2 activation, as well as the regulation of inhibi-
tory κB kinase-β (IKK-β) activity responsible for IκB-α
degradation and NF-κB nuclear translocation [26-29].
Moreover, we have recently shown that S-nitrosylation
of IKK-β plays a role in the modulatory influence of pep-
tide hormone, ghrelin, on the gastric mucosal inflamma-
tory responses to H. pylori [11,17,30].
As gastric mucosal responses to H. pylori are associ-
ated with MAPK/JNK, p38 and ERK activation, while
the modulatory influence of ghrelin is reflected in the
inhibition of JNK and p38, but not the ERK, in this study
we investigated the influence of H. pylori LPS and ghre-
lin on the processes affected by MAPK/ERK activation.
Our results demonstrate that the LPS-induced ERK acti-
vation is of critical significance to up-regulation in gas-
tric mucosal PGE2 and NO generation at the level of
IKK-β and cPLA2 activation, and that ghrelin counters
these proinflammatory consequences of the LPS through
Src/Akt-dependent S-nitrosylation.
2. Materials and Methods
2.1. Gastric Mucosal Cell Incubation
The gastric mucosal cells, collected form freshly dissected
rat stomachs by scraping the mucosa with a blunt spatula,
were suspended in five volumes of ice-cold Dulbecco’s
modified (Gibco) Eagle’s minimal essential medium
(DMEM), supplemented with fungizone (50 µg/ml), peni-
cillin (50 U/ml), streptomycin (50 µg/ml), and 10% fetal
calf serum. After gentle trituration with a syringe, the dis-
persed cells were settled by centrifugation, and resuspended
in the medium to a concentration of 2 × 107 cell/ml [31].
Cell aliquots (1 ml) were then transferred to DMEM in
culture dishes and incubated under 95% O2 - 5% CO2
atmosphere at 37˚C for up to8 h in the presence of 0 -
100 ng/ml of H. pylori LPS [6]. H. pylori used for LPS
preparation was cultured from clinical isolates obtained
from ATCC No. 4350 [6]. In the experiments evaluating
the effect of ghrelin (rat, Sigma), JNK inhibitor, SP600125,
p38 MAPK inhibitor, SB202190, ERK1/2 inhibitor,
PD98059, Akt inhibitor, SH-5, Src inhibitor, PP2, Raf-1
kinase inhibitor, NF-κB inhibitor, PPM-18 (Calbiochem),
and ascorbate (Sigma), the cells were first preincubated
for 30 min with the indicated dose of the agent or vehicle
before the addition of the LPS. The viability of cell
preparations before and during the experimentation, as-
sessed by Trypan blue dye exclusion assay [6], was
greater than 97%.
2.2. COX-2 and iNOS Activity Assay
For measurements of COX-2 activity, the gastric mucosal
cells from the control and various experimental treatments
were settled by centrifugation, rinsed with phosphate-
buffered saline, and homogenized in 0.3 ml of cold sam-
ple buffer containing 0.1 M Tris-HCl, pH 7.8, and 1 mM
EDTA, centrifuged at 12,000 × g for 10 min at 4˚C, and
the supernatant collected [5]. The COX-2 activity in 40
µl sample aliquots of the resulting supernatant was meas-
ured using COX activity assay kit (Cayman) in the ab-
sence and the presence of COX-1 inhibition (SC-560), by
monitoring the appearance of oxidized TMPD at 590 nm.
The activity of iNOS was measured by monitoring the
conversion of L-[3H] arginine to L-[3H] citrulline using
NOS-detect kit (Stratagene). The gastric mucosal cells
from the control and experimental treatments were ho-
mogenized in a sample buffer containing 10 mM EDTA
and centrifuged. The aliquots of the resulting supernatant
were incubated for 30 min at 25˚C in the presence of 50
µCi/ml of L-[3H] arginine, 10 mM NAPDH, 5 µM tetra-
hydrobiopterin, and 50 mM Tris-HCl buffer, pH 7.4, in a
final volume of 250 µl. Following addition of stop buffer
and Dowex-50 W (Na+) resin, the mixtures were trans-
ferred to spin cups, centrifuged and the formed L-[3H]
citrulline contained in the flow through was quantified by
scintillation counting [6].
2.3. cPLA2 Activity Assay
The cPLA2 activity measurements were carried out using
cPLA2 assay kit (Cayman) with thioarachidonoylphos-
phatidylcholine as substrate. The gastric mucosal cells
from the control and experimental treatments were set-
tled by centrifugation, homogenized in 1 ml of 50 mM
HEPES buffer, pH 7.4, containing 1 mM EDTA, and
centrifuged at 10,000 × g for 15 min at 4˚C [29]. The su-
pernatants were then filtered through an Amicon YM30
filter concentrators (m.w. cut-off 30 KDa) to remove any
contamination with secretory PLA2, followed by 15 min
incubation with 5 µM calcium-independent PLA2 inhibi-

B. L. SLOMIANY, A. SLOMIANY 23
tor, bromoenol lactone, and the aliquots (10 µl) of such
prepared cell lysates were subjected to cPLA2 assay ac-
cording to manufacturer’s instruction.
2.4. IκB Kinase Activity Assay
To measure the IKK-β activity we utilized the ELISA-
based detection kit, K-LISA (Calbiochem). The GST-
IκB-α 50-amino acid peptide that includes the Ser32 and
Ser36 of IκB-α phosphorylation sites was used as a sub-
strate [32]. The gastric mucosal cell cytosolic extracts
were incubated a glutathione-coated 96-well plate with
GST-tagged IκB-α at room temperature for 30 min, and
the phosphorylated GST-IκB-α substrate was detected
using anti-phospho IκB-α (Ser32/Ser36) as first antibody,
followed by horseradish peroxidase-conjugated secon-
dary antibody. Following washing the retained complex
was probed TMB reagent for spectrophotometric quanti-
fication [11].
2.5. cPLA2 and IKK-β Protein S-Nitrosylation
Assay
Detection of cPLA2 and IKK-β protein S-nitrosylation
was carried out utilizing a biotin switch method for pro-
tein S-nitrosylation [33,34]. The gastric mucosal cells
were treated with ghrelin (0.5 µg/ml), or ERK inhibitor,
PD 98059 (30 µM) + ghrelin, or Akt inhibitor, SH-5 (20
µM) + ghrelin, and incubated for 1h in the presence of
100ng/ml of H. pylori LPS. The cells were collected by
centrifugation at 500 × g for 5 min, lysed in 0.2 ml of
HEN lysis buffer (250 mM HEPES, 1 mM EDTA, 0.1
mM neocuprin, pH 7.7), and the unnitrosylated thiol
groups were blocked with S-methyl methanethiosul-
fonate reagent at 50˚C for 20 min [34]. The proteins were
precipitated with acetone, resuspended in 0.2 ml of HEN
buffer containing 1% SDS, and subjected to targeted ni-
trothiol group reduction with sodium ascorbate (100 mM).
The free thiols were then labeled with biotin and the
biotinylated proteins were recovered on streptavidin beads.
The formed streptavidin bead-protein complex was washed
with neutralization buffer, and the bound proteins were
dissociated from streptavidin beads with 50 µl of elution
buffer (20 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH
7.7) containing 1% 2-mercaptoethanol [34]. The obtained
proteins were then analyzed by Western blotting.
2.6. Nuclear Protein Extraction
The aliquots of gastric mucosal cell suspension from the
control and various experimental conditions were settled
by centrifugation at 500 × g for 5 min, rinsed with phos-
phate-buffered saline, and lysed by incubation for 10 min
on ice in the lysis buffer, containing 10 mM HEPES, pH
7.9, 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 1
mM dithiothreitol, and 0.5 mM PMSF [35]. Following
centrifugation at 12,000 × g for 10 min at 4˚C, the super-
natant was subjected to centrifugation at 100,000 × g for
1h and the obtained soluble fraction was used as source
of cytosolic extract [11]. The pellets, from 12,000 × g
centrifugation, containing crude nuclei were suspended
for 20 min at 4˚C in the extraction buffer, containing 20
mM HEPES, pH 7.9, 25% glycerol, 400 mM NaCl, 1.5
mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, and 1
mM PMSF. The samples were centrifuged at 15,000 × g
for 10 min at 4˚C, and the supernatants containing nu-
clear extracts were collected and stored at –70˚C until
use.
2.7. Immunoblot Analysis
The gastric mucosal cells from the control and experi-
mental treatments were collected by centrifugation and
resuspended for 30 min in ice-cold lysis buffer (20 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Tri-
ton X-100, 2 mM EDTA, 1 mM sodium orthovanadate, 4
mM sodium pyrophosphate, 1 mM PMSF, and 1 mM
NaF), containing 1 µg/ml leupeptin and 1 µg/ml pepstatin
[5]. Following brief sonication, the lysates were centri-
fuged at 12,000 × g for 10 min, and the supernatants were
collected and normalized with respect to protein concen-
tration using BCA protein assay kit (Pierce). The samples,
including those subjected to biotin switch procedure,
were then resuspended in loading buffer, boiled for 5 min,
and subjected to SDS-PAGE using 40 µg protein/lane.
The separated proteins were transferred onto nitrocellu-
lose membranes, blocked for 1 h with 5% skim milk in
Tris-buffered Tween (20 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 0.1% Tween-20), and probed with specific anti-
bodies directed against IκΒ-α, COX-2 and iNOS (Cal-
biochem), NF-κB p65 and IKK-β (EMD Millipore), and
phospho-IKK-β, cPLA2 and phospho-cPLA2 (Cell Sig-
naling) Antibodies directed against ERK1/2 and phos-
pho-ERK1/2 MAPK were from Calbiochem, whereas
ati-β-actin from Sigma.
2.8. Data Analysis
All experiments were carried out using duplicate sampling,
and the results are expressed as means ± SD. Analysis of
variance (ANOVA) and nonparametric Kruskal-Wallis
tests were used to determine significance. Any difference
detected was evaluated by means of post hoc Bonferroni
test, and the significance level was set at P < 0.05.
3. Results
In our previous study, we reported that ghrelin modulates
H. pylori LPS-elicited induction in gastric mucosal COX-2
and iNOS expression by affecting p38 MAPK/ATF-2
and IKK-β/NF-κB activation pathways [17]. Indeed, as
shown in Figure 1, exposure of rat gastric mucosal cells

B. L. SLOMIANY, A. SLOMIANY 24
to H. pylori LPS lead to a significant induction in the
level of COX-2 and iNOS proteins (Figure 1(b)), accom-
panied by a marked increase in the mucosal cell COX-2
and iNOS enzymatic activities (Figure 1(c)). Further,
preincubation with ghrelin lead to suppression of the
LPS-induced iNOS and COX-2 activities. However,
while the effect of ghrelin on iNOS activity was also
manifested in a marked inhibition of the iNOS protein
expression, less apparent change was observed in the
expression of COX-2 protein (Figure 1(b)).
(a)
(b)
(c)
Figure 1. Effect of ghrelin (Gh) on H. pylori LPS-induced
changes in the expression and activity of COX-2 and iNOS
proteins in gastric mucosal cells. The cells were treated with
the LPS at 100 ng/ml or Gh at 0.5 µg/ml + LPS, and incu-
bated for 8 h. Cell lysates were analyzed by western blotting
with specific antibodies (a) for COX2 and iNOS; their pro-
tein expression (b); and the enzymatic activity (c). Actin bl ot
shows equal lane load. The data repre sent the mean ± SD of
four experiments. (*P < 0.05 compared with that of control.
**P < 0.05 compared with that of LPS.)
Moreover, since induction of iNOS and COX-2 en-
zymes in response to LPS is controlled primarily by fac-
tors operating at the level of transcriptional activation
[2,7-9,17], we assessed the expression of COX-2 and
iNOS proteins in the presence of the inhibitors of MAPK
and NF-κB activation. The results revealed that H. pylori
LPS-induced expression of COX-2 protein showed sus-
ceptibility to inhibition by the p38 inhibitor, SB202190,
whereas the LPS-induced expression of iNOS protein
was subject to suppression by the inhibitor of NF-κB,
PPM-18, as well as the inhibitor of ERK1/2, PD98059
(Figure 2). These results thus confirm the existence of an
intimate relationship between the pathways of MAPK
and NF-κB activation for the induction of proinflamma-
tory iNOS and COX-2 expression in response to H. py-
lori colonization.
As gastric mucosal responses to H. pylori are associ-
ated with MAPK/JNK, p38 and ERK activation, while
the modulatory influence of ghrelin is reflected in the
inhibition of JNK and p38 phosphorylation but not that
of ERK [17], we examined the influence of the LPS and
(a)
(b)
Figure 2. Effect of MAPK and NF-  B inhibitors on H. pylori
LPS-induced changes in COX-2 and iNOS protein expres-
sion in gastric mucosal cells. The cells were treated with 30
µM PD98059 (PD), 10 µM SP600125 (SP), 20 µM SB202190
(SB), or 15 µM PPM-18 (PPM), and incubated for 8 h with
the LPS at 100 ng/ml. Cell lysates were analyzed by western
blotting for COX-2 and iNOS proteins (a) and their protein
expression (b) are normalized to  actin. The data represent
the mean ± SD of four experiments. (*P < 0.05 compared
with that of LPS.)

B. L. SLOMIANY, A. SLOMIANY 25
ghrelin on the processes affected by ERK activation. The
results revealed that the LPS effect, manifested in ERK
activation and further enhanced by ghrelin, was associ-
ated with IKK-β phosphorylation, and the inhibitory ef-
fect of PD98059 on ERK phosphorylation was also re-
flected in a reduced phosphorylation of IKK-β Figure 3).
However, we found that in contrast to the LPS, the effect
of ghrelin on IKK-β was associated with the inhibition of
the LPS-induced IκB-α degradation in the cytosol and a
marked decrease in the LPS-induced translocation of p65
NF-κB into nucleus (Figure 4). As a consequence, this
effect of ghrelin is manifested in the suppression of iNOS
gene induction at the level of NF-κB activation.
(a)
(b)
Figure 3. Effect of ghrelin (Gh) on H. pylori LPS-induced
changes in gastri c mucosal cell ERK and IKK-  phosphoryla-
tion. The cells, preincubated with 0 or 0.5 µg/ml ghrelin, or
30 µM PD98059 (PD) + Gh, were incubated for 30 min with
the LPS at 100 ng/ml. Cell lysates were analyzed by western
blotting for total and phosphorylated ERK and IKK-  with
respective specific antibodies (a); and the relative densities
of phosphorylated (pERK and pIKK  proteins (b) are ex-
pressed as fold of control. Actin blot show s equal lane load.
The data represent the mean ± SD of four experiments. (*P
< 0.05 compared with that of control. **P < 0.05 compared
with that of LPS. ***P < 0.05 compared with that of Gh 
LPS.)
Hence, in further assessment of the role of ERK acti-
vation in modulation of COX-2 and iNOS expression by
ghrelin, we analyzed the changes in enzymatic activity of
IKK-β in gastric mucosal cells exposed to H. pylori LPS.
The results revealed that the LPS-induced up-regulation
in IKK-β activity was subject to suppression by ghrelin
as well as the inhibitors of ERK activation, PD98059 and
Raf-1kinase inhibitor, and the effects were additive (Fig-
ure 5). Moreover, the effect of ghrelin on the LPS-in-
duced up-regulation in IKK-β activity was susceptible to
reversal by the inhibitors of Src/Akt pathway, PP2 and
SH-5. The Src/Akt inhibitors, however, produced no dis-
cernible effect on the extent of the LPS-induced IKK-β

Figure 4. Effect of ghrelin (Gh) on H. pylori LPS-induced
IKK-  phosphorylation, I  B-  degradation, and p65 NF-
 B nuclear translocation in gastric mucosal cells. The cells,
preincubated with 0 or 0.5 µg/ml Gh, were incubated for 30
min with the LPS at 100 ng/ml. Cell lysates were analyzed
by western blotting for I  B-  and pIKK  while the level of
p65 NF-  B was assessed in the nuclear fraction. Actin blot
shows equal line load. The immunoblots shown are repre-
sentative of three experiments. 


Figure 5. Effect of Src, Akt, Raf-1 kinase, and ERK inhibi-
tors on the ghrelin (Gh)-induced changes in the expression
of IKK-  activity in gastric mucosal cells exposed to H. py-
lori LPS. The cells, preincubated with 30 µM PP2, 20 µM
SH-5 (SH), 30 µM PD98059 (PD), or 10 µM Raf-1 kinase
inhibitor (Rf), were treated with Gh at 0.5 µg/ml and incu-
bated for 30 min in the presence of 100 ng/ml of LPS. Val-
ues represent the mean ± SD of five experiments. (*P < 0.05
compared with that of control. **P < 0.05 compared with
***
that of LPS. P < 0.05 compared with that of Gh  LPS.)

B. L. SLOMIANY, A. SLOMIANY 26
activity. This indicates that the countering effect of ghrelin
matory consequence of COX-2 in-
du
on H. pylori LPS-elicited up-regulation in gastric muco-
sal IKK-β activation, and the suppression of proinflam-
matory COX-2 and iNOS enzymes induction, occurs
with the involvement of pathways regulated by ERK and
Src/Akt activation.
As the proinflam
ction by H. pylori is the massive up-regulation in gas-
tric mucosal prostaglandin production, we have turned
our attention to the processes associated with the release
of AA from membrane phospholipids by the action of
cPLA2. [21]. Following on the documented involvement
of ERK in the processes of cPLA2 activation, we ana-
lyzed the effect of H. pylori LPS and ghrelin on gastric
mucosal cPLA2 phosphorylation. The results revealed
that the LPS-induced ERK phosphorylation was associ-
ated with a marked increase in cPLA2 phosphorylation,
and that the phosphorylation of both enzymes, and cPLA2
in particular, was further enhanced in the presence of
ghrelin (Figure 6).
(a)
(b)
Figure 6. Effect of ghrelin H. pylori LPS-induced
LPS.)
r
(Gh) on
cPLA2 and ERK phosphorylation in gastric mucosal cells.
The cells, preincubated with 0 or 0.5 µg/ml ghrelin, were
incubated for 30 min with the LPS at 100 ng/ml, and the
lysates were analyzed by western blotting for total and
phosphorylated cPLA2 and ERK and with respective spe-
cific antibodies (a); and the relative densities of phosphory-
lated (pcPLA2 and pERK proteins (b) are expressed as fold
of control. Actin blot shows equal lane load. The data rep-
resent the mean ± SD of four experiments. (*P < 0.05 com-
pared with that of control. **P < 0.05 compared with that of
Moreover, by measuring the mucosal cell cPLA2 en-
zymatic activity, we found that the LPS-induced incease
in the enzyme protein phosphorylation was reflected in
up-regulation in cPLA2 activity, and that this effect of the
LPS was susceptible to suppression by ghrelin as well as
the inhibitors of ERK, PD98059 and Raf-1 inhibitor
(Figure 7). The countering effect of ghrelin on the LPS-
induced up-regulation in cPLA2 activity, furthermore,
was subject to reversal by the inhibitors of Src/Akt
pathway, PP2 and SH-5, but neither inhibitor produced
any discernible alteration in the LPS-induced cPLA2 ac-
tivity. These results thus point to the role of ERK and
Src/Akt pathways in the modulation of cPLA2 activation
by ghrelin in response to H. pylori LPS.
In further assessment of factors that influence the
modulatory action of ghrelin on H. pylori LPS-induced
up-regulation in gastric mucosal NO and PGE2 produc-
tion, we investigated the effect of nitrosothiols reducing
agent, ascorbate, on the activity of IKK-β and cPLA2
enzymes. The results revealed that, while preincubation
with ascorbate produced no discernible result on the ex-
tent of the LPS-induced activation, the agent elicited a
marked relieve in the inhibitory effect ghrelin on the
LPS-induced IKK-β and cPLA2 activity (Figure 8).
However, ascorbate produced only negligible changes in
the effect ERK inhibitor, PD98059, on the LPS-induced
activity of both enzymes. Therefore, to ascertain further
the relationship between the processes of IKK-β and
cPLA2 activation by H. pylori LPS, and the modulatory
influence of ghrelin, we examined the patterns of IKK-β
and cPLA2 S-nitros ylation in conjunction with the phos-
phorylation requirements. We observed that gastric mu-
cosal cells exposed to H. pylori LPS showed a substantial
Figure 7. Effect of Src, Akt, Raf-1 kinase, and ERK inhib
tors on the ghrelin (Gh)-induced changes in the expressioi- n
of cPLA2 activity in gastric mucosal cells exposed to H. py-
lori LPS. The cells, preincubated with 30 µM PP2, 20 µM
SH-5 (SH), 30 µM PD98059 (PD), or 10 µM Raf-1 kinase
inhibitor (Rf), were treated with Gh at 0.5 µg/ml and incu-
bated for 30 min in the presence of 100 ng/ml of LPS. Val-
ues represent the mean ± SD of five experiments. (*P < 0.05
compared with that of control. **P < 0.05 compared with
that of LPS. ***P < 0.05 compared with that of Gh  LPS.)

B. L. SLOMIANY, A. SLOMIANY 27
increase in IKK-β and cPLA2 phosphorylation, while
the countering effect of ghrelin on the LPS-induced
astric mucosa by H. pylori or stimula-
cosal cells with Hggers
activation of the enzymes was manifested in a marked
increase in S-nitrosylation of the both enzymes (Figure
9). Furthermore, preincubation with Akt inhibitor, SH-
5, resulted in the blockage of the ghrelin-induced S-ni-
trosylation of IKK-β and cPLA2, but it had no effect on
phosphorylation of the proteins, whereas ERK inhibitor,
PD98059, caused the blockage in IKK-β and cPLA2 pho-
sphorylation as well as S-nitrosylation. Thus, ghrelin
affects the phosphorylation as well as S-nitrosylation of
IKK-β and cPLA2, and that in both cases the phosphory-
lation event is a prerequisite for S-nitrosylation. Further,
the data suggest that H. pylori LPS-induced ERK activa-
tion plays a critical role in up-regulation of gastric mu-
cosal nitric oxide and prostaglandin production at the
level of IKK-β and cPLA2 activation, and that ghrelin
counters these untoward consequences of the LPS through
Src/Akt-dependent S-nitrosylation.
4. Discussion
Colonization of g
tion of gastric mu. pylori LPS tri
the release of excessive amounts of NO and PGE2, the
overproduction of which is not only detrimental to bacte-
rial survival but also cause tissue injury, and hence in-
crease the risk of gastric disease [1,2,4-6]. The induction
of iNOS and COX-2 genes responsible for rapid up-
regulation in NO and PGE2 generation is the result of
stimulation by the LPS of gastric mucosal TLR-4 and the
activation of cognate transcription factors as well as each
of the three MAPK subtypes; JNK, p38 and ERK [2,17].
While the LPS-elicited induction in iNOS expression
Figure 8. Effect of ascorbate on the LPS and ghrelin (Gh)
induced changes in the expression of IKK-  and cPLA
-
2
activities in gastric mucosal cells. The cells, preincubated
with 300 µM ascorbate (As) or 30 µM PD98059 (PD), were
treated with 0.5 µg/ml Gh and incubated for 30 min in the
presence of 100 ng/ml LPS. Values represent the mean ± SD
of five experiments. (*P < 0.05 compared with that of con-
trol. **P < 0.05 compared with that of LPS. ***P < 0.05 com-
pared with that of Gh  LPS.)
Figure 9. Effect of H. pylori LPS and ghrelin (Gh) on LA2
and IKK-  protein S-nitrosylation in gastric mucosal cells.
ranscriptional factor, NF-κB,
nd the MAPK/JNK and p38 activation is associated
ori
LP
cP
The cells, preincubated for 30 min with Gh at 0.5 µg/ml, or
30 PD98050 (PD) + Gh, or 20 µM SH-5 (SH) + Gh, and in-
cubated for 1 h in the presence of 100 ng/ml LPS. A portion
of the cell lysates was processed by biotin switch procedure
for protein S-nitrosylation and, along with the reminder of
the lysates, analyzed by western blotting for total and
phosphorylated cPLA2 and IKK-  proteins with respective
specific antibodies. The immunoblots shown are represen-
tative of three experiments.
appears to be regulated by t
a
with the induction in COX-2 expression through tran-
scription factor, AP-1 activation, the data on the func-
tional significance of LPS-induced MAPK/ERK activa-
tion remain at variance [9-12,16,17]. Depending on the
cell type, the MAPK/ERK activation has been implicated
in the control of COX-2 expression through transcription
factors, C/EBP and CREB activation, as well as the en-
hancement in NF-κB nuclear translocation associated
with the induction in iNOS expression [14,15,18-20].
Accordingly, in the present study, we explored the in-
volvement of MAPK/ERK in the regulation of H. pyl
S-induced gastric mucosal expression of COX-2 and
iNOS. Our data, obtained with rat gastric mucosal cells,
revealed that ERK activation by the LPS was associated
with a marked increase in the phosphorylation of IKK-β
and cPLA2, and reflected in the upsurge in NF-κB nuclear
translocation, induction in COX-2 and iNOS expression,
and up-regulation in cPLA2 activity. Moreover, the LPS-
induced up-regulation in IKK-β and cPLA2 activity was
susceptible to suppression by the inhibitors of ERK pho-
sphorylation, PD98059 and Raf-1 inhibitor, thus sup-
porting the role of ERK in the processes of cPLA2 acti-
vation for the increase in AA release for COX-2-medi-
ated prostaglandin synthesis, as well as the NF-κB-de-
pendent induction of iNOS expression for the increase in
NO generation. Indeed, the literature data indicate that
cPLA2 activation requires MAPK/ERK-mediated en-
zyme phosphorylation on the critical Ser505 residue that

B. L. SLOMIANY, A. SLOMIANY 28
plays a crucial role in Ca2+-dependent translocation of
cPLA2 form cytosol to membrane to gain access to pho-
spholipid substrates [21,36]. Likewise, the ability of ERK
to exert a stimulatory effect on IKK/NF-κB activation
through Raf-1/MEK/ERK signaling cascade has been
implicated in the regulation of adenosine A1 receptor
mediated activation of NF-κB in human lymphocytic
cells [19].
Further, we found that, while the LPS-induced phos-
phorylation of ERK, IKK-β and cPLAwas enhanced by
pr
ty of substrates, and the signaling through Src/
A
and cPLA activation by
th
n as to the critical role of
M
2
eincubation with gastric hormone, ghrelin, the modu-
latory effect of the hormone was reflected in the suppres-
sion of IKK-β and cPLA2 activity, as well as that of
iNOS and COX-2. The effect of ghrelin on iNOS was
manifested in a marked decline of the enzyme protein
expression, and associated with the inhibition of the
LPS-induced IκB-α degradation and a decrease in NF-κB
nuclear translocation, while the effect on COX-2 was
primarily manifested in the suppression of the LPS-in-
duced up-regulation in COX-2 activity. Interestingly, NO
stimulation through iNOS induction has been linked to
COX-2 activation through S-nitrosylation and the in-
crease in PGE2 production [9,26], and we have recently
shown that ghrelin suppression of H. pylori LPS-induced
COX-2 S-nitrosylation results in the inhibition of PGE2
generation [5]. Moreover, following our earlier leads as
to the involvement of cNOS in the mechanism of ghrelin
action [6,11], we revealed that the countering effect of
ghrelin on the LPS-induced up-regulation in the activity
of IKK-βand cLPA2 enzymes was susceptible to sup-
pression by the inhibitors of cNOS activation through
phosphorylation, an Akt inhibitor, SH-5 and Src inhibitor,
PP2. The Src/Akt inhibitors, however, produced no dis-
cernible alteration in the LPS-induced activity of IKK-β
and cPLA2 enzymes. These data and the finding that the
LPS-induced up-regulation in the activities of IKK-β and
cPLA2 was also susceptible to suppression by the inhibi-
tors of ERK activation, PD98059 and Raf-1, provide
strong indication as to the role of ERK and Src/Akt
pathways in the mediation of modulatory influence of
ghrelin on the processes associated with up-regulation in
gastric mucosal PGE2 and NO generation in response to
H. pylori.
While as an upstream kinase, cSrc phosphorylates a
wide varie
kt pathway is known to occupy a central stage in the
receptor (GHS-R)-mediated responses to ghrelin stimula-
tion [6,11,37-39], it is becoming increasingly apparent
that the hormone is also capable of exerting its modula-
tory influence through the process of protein S-nitrosy-
lation. Indeed, the induction IKK-β S-nitrosylation by
ghrelin exerts the inhibitory effect on the extent of IκB-
α degradation, causing suppression in NF-κB nuclear
translocation and resulting in the repression of iNOS
gene induction [11,31]. Moreover, ghrelin has been im-
plicated in the modulation of S-nitrosylation-dependent
activation COX-2 and cPLA2 enzymes, thus affecting the
processes of PGE2 generation [11,27,29,30]. Therefore,
in further assessment of the modulatory action of ghrelin
on H. pylori LPS-induced up-regulation in gastric muco-
sal generation of PGE2 and NO, we evaluated the effect
of nitrosothiol reducing agent, ascorbate on the activity
of IKK-β and cPLA2. We found that while ascorbate elic-
ited a marked relieve in the inhibitory effect of ghrelin on
the LPS-induced IKK-β and cPLA2 activation, the agent
produced only negligible changes in the effect of ERK
inhibitor, PD98059, on the LPS-induced activity of both
enzymes. Hence, consistent with the above findings and
considering the fact that the changes in activity of both
enzymes in the presence of ghrelin was susceptible to
suppression by the inhibitors of cNOS activation, we
concluded that the countering effect of ghrelin on H. py-
lori LPS-induced activation of IKK-β and cPLA2 are in-
timately linked to the events of cNOS-dependent S-ni-
trosylation of these enzymes.
Consequently, to ascertain further the relationship be-
tween the processes of IKK-β2
e LPS, and the modulatory action of ghrelin, we exam-
ined the patterns of IKK-β and cPLA2 S-nitrosylation in
conjunction with the phosphorylation requirements in the
presence of the inhibitors of cNOS and ERK. We ob-
served that the mucosal cells exposed to the LPS showed
a marked increase in IKK-β and cPLA2 phosphorylation,
while the countering effect of ghrelin was associated
with the increased phosphorylation as well as S-nitrosy-
lation of the enzymes. Furthermore, preincubation with
the inhibitor of cNOS activation, SH-5, lead to the
blockage in ghrelin-induced S-nitrosylation of IKK-β and
cPLA2, but had no effect on phosphorylation of the en-
zymes, whereas ERK inhibitor, PD98059, caused the
blockage in IKK-β and cPLA2 phosphorylation as well as
S-nitrosylation. This indicated that the phosphorylation
event is a prerequisite for the IKK-β and cPLA2 protein
S-nitrosylation. Together, these findings suggest that the
activation MAPK/ERK by H. pylori LPS plays a pivotal
role in up-regulation in gastric mucosal PGE2 and NO
generation at the level of IKK-β and cPLA2 activation
through phosphorylation, and that ghrelin counters these
proinflammatory consequences of the LPS through Src/
Akt-mediated and cNOS-dependent S-nitrosylation of the
IKK-β and cPLA2 proteins.
In conclusion, the data present in this report add fur-
ther support to our assertio
APK/ERK/JNK and p38 signaling cascades in media-
tion of gastric mucosal inflammatory responses to H.
pylori LPS While the LPS-elicited induction in COX-2
expression relays primarily on JNK/p38-dependent acti-
vation of transcription factor AP-1 [17], the ERK

B. L. SLOMIANY, A. SLOMIANY 29
Figure 10. Schematic representation of the modulatory mechanism of ghrelin action in countering the excessive gastric mu-
cosal NO and PGE2 generation in response to H. pylori LPS. Binding of the LPS to Toll-like receptor 4 (TLR4)/MD2 tri
vents leading to the activation of cPLA and IKK-β thus
ENCES
ated Gastritis May
Represent an o Develop Gastric
ggers
the activation of JNK, p38 and ERK1/2 MAPKs, and nuclear translocation of transcription factors involved in the inductio n
of COX-2 (AP-1, CREB and C/EBP), and iNOS (NF-
 B) genes transcription. While JNK and p38 MAPKs are involved in the
regulation of AP-1 assembly [17], activation of ERK by the LPS leads to phosphorylation and activation of IKK-β and cPLA2,
which trigger up-regulation in arachidonic acid (AA) re lease and the upsurge in NF-κB nuclear translocation. The induction
in iNOS and massive rise in NO leads to COX-2 activation through S-nitrosylation that results in the excessive PGE2 genera-
tion. Engagement of the growth hormone secretagogue receptor (GHSR) by ghrelin leads to the inhibition of C/EBP and
p38/JNK-mediated AP-1 activation, and hence results in the reduced COX-2 expression. Moreover, the effect of ghrelin is
reflected in further enhancement in the LPS-induced ERK activation, and up-regulation in Src/Akt-dependent cNOS phos-
phorylation that leads to the inhibition of IKK-  and cPLA2 activation by cNOS–mediated S-nitrosy lation. This results in the
repression of iNOS gene induction and the inhibition of COX-2 activation through iNOS-dependent S-nitr osy lation, as well as
the suppression of AA release. AP-1, activator protein-1; ATF-2, activating transcription factor-2; CREB, cAMP response
element binding protein; C/EBP, CCAAT/enhancer binding protein; PGH2, prostaglandin H2.
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