in the amplification of Helicobacter pylori-elicited induction in ...(TLR-4), which then through a series of downstream effectors, causes the activation of transcriptional factors

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Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)

For citation purposes: Slomiany BL, Slomiany A. Role of epidermal growth factor receptor transactivation in the amplification of Helicobacter pylori-elicited induction in gastric mucosal expression of cyclooxygenase-2 and inducible nitric oxide synthase. OA Inflammation 2013 Apr 01;1(1):1. Co

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Cell

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Role of epidermal growth factor receptor transactivation in the amplification of Helicobacter pylori-elicited

induction in gastric mucosal expression of cyclooxygenase-2 and inducible nitric oxide synthase

BL Slomiany*, A Slomiany

AbstractIntroductionIn this study, we report on the role of epidermal growth factor receptor (EGFR) transactivation in H. pylori l-ipopolysaccharide (LPS)-elicited in-duction in gastric mucosal expressi-on of COX-2 and iNOS.Materials and methodsWe aimed to demonstrate that the LPS-induced p38 activation along w-ith MMPs are of critical significance to EGFR transactivation that leads to up-regulation in the activation of E-RK signalling cascade, amplification in iNOS and COX-2 induction, and c-onsequently to the excessive increa-se in gastric mucosal PGE2 and NO generation.ResultsEGFR transactivation results in the amplification of the LPS-induced ER-K phosphorylation and up-regulatio-n in ERK-mediated IKK-β activation for the enhanced induction of NF-κB-dependent expression of iNOS. T-he rise in iNOS-dependent NO gene-ration, in turn, leads to an up-regul-ation in COX-2 activation through S-nitrosylation and excessive PGE2 production.ConclusionTaken together, our data

provide strong indications of the in-volvement of EGFR transactivation in the amplification of ERK signalling cascade associated with up-regula-tion in gastric mucosal induction of iNOS and COX-2, and excessive NO and PGE2 generation in response to H. pylori.

IntroductionInfection with Helicobacter pylori is recognized as a primary factor in the aetiology of gastric disease, and results in the excessive generation of prostaglandin (PGE2) and nitric ox-ide (NO) triggered by disturbances in cyclooxygenase (COX). Nitric oxide synthase (NOS) isozyme systems are considered to be of major importance in defining the extent of gastric mu-cosal inflammatory involvement1–6. Studies on the signalling events un-derlying the up-regulation of PGE2 and NO generation indicate that H. pylori cell wall lipopolysaccharide (LPS), like that of other Gram-nega-tive bacteria, is capable of triggering the stimulation of Toll-like receptor-4 (TLR-4), which then through a series of downstream effectors, causes the activation of transcriptional factors that exert control over NOS and COX-2 gene expression2,7–9.

The engagement of TLR-4 by LPS, moreover, is known to elicit the acti-vation 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 fac-tor activation through phosphoryla-tion10–13. Indeed, we have recently

shown that stimulation of gastric mucosal cells with H. pylori LPS elic-its the activation of JNK, p38 and ERK and have linked the involvement of JNK/p38 in the transcription factor AP-1 activation, as well as provided evidence for the role of ERK activa-tion in the regulation of factors linked to the induction of COX-2 and induci-ble NOS (iNOS) expression for the in-crease in PGE2 and NO generation9,14.

Moreover, literature indicates that LPS-induced activation of MAPK cas-cade is also associated with epider-mal growth factor receptor (EGFR) transactivation15–17. EGFR is a trans-membrane glycosylated protein with intrinsic tyrosine kinase activity that controls a wide variety of cell functions that are of significance to mucosal defence and repair. These include processes such as cellular proliferation, differentiation, migra-tion and modulation of apoptosis18,19. Studies indicate that in addition to direct EGFR activation by its cognate EGF peptide ligand, transactivation of EGFR requires an extracellular matrix metalloproteinase (MMP)-mediated shedding of heparin-binding EGF-like growth factor (HB-EGF) and its sub-sequent binding to the receptor19,20. Furthermore, the crosstalk between TLR4 and EGFR signalling systems has been linked to rapid changes in the extent of mucosal inflammation with bacterial invasion21,22. In partic-ular, there are strong indications that p38 MAPK activation plays an impor-tant role in MMP-2 and MMP-9 acti-vation and that EGFR transactivation results in up-regulation in ERK MAPK pathway stimulation23–26.

* Corresponding authorEmail: [email protected]

Research Centre, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103 – 2400, USA

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For citation purposes: Slomiany BL, Slomiany A. Role of epidermal growth factor receptor transactivation in the amplification of Helicobacter pylori-elicited induction in gastric mucosal expression of cyclooxygenase-2 and inducible nitric oxide synthase. OA Inflammation 2013 Apr 01;1(1):1. Com

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EGFR transactivation assayMeasurement of EGFR transacti-vation was conducted with Phos-phoDetect Elisa kit (Calbiochem). The gastric mucosal cells from the control and various experimental conditions were washed twice with phosphate-buffered saline, treated with the receptor extraction buffer and centrifuged at 1500× g for 10 min at 4°C. The supernatant was then incubated at room temperature for 2 h with anti-EGFR antibody, followed by 30-minute incubation with pro-tein A agarose and centrifugation28. After washing, the pellet was sus-pended in kinase reaction buffer, and 100 µL aliquots were used for EGFR phosphotyrosine assay following the manufacturer’s instructions.

IκB kinase activity assayTo measure the inhibitory κB kinase-β (IKK-β) activity, we utilized the ELI-SA-based detection kit, K-LISA (Cal-biochem). The GST-IκB-α50-amino acid peptide that includes the Ser32 and Ser36 of IκB-α phosphorylation sites was used as a substrate27,29. The gastric mucosal cell cytosolic extracts were incubated with 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 horserad-ish peroxidase-conjugated second-ary antibody. Following washing, the retained complex was probed with 3,3’,5,5’-tetramethylbenzidine rea-gent for spectrophotometric quanti-fication.

Immunoblot analysisThe gastric mucosal cells from the control and experimental 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 py-rophosphate, 1 mM PMSF and 1 mM

spectrum MMPinhibitor, GM6001 (Calbiochem), the cells were first pr-eincubated for 30 min with the indi-cated dose of the agent or vehicle before the addition of the LPS. The viability of cell preparations before and during the experimentation, ass-essed by Trypan blue dye exclusion assay6, was greater than 97%.

iNOS activity assayThe activity of iNOS enzyme in gas-tric mucosal cells 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 con-trol and experimental treatments were homogenized in a sample buffer containing 10 mM EDTA and centrifuged. The aliquots of the re-sulting 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 tetrahydrobiop-terin and 50 mM Tris-HCl buffer, pH 7.4, in a final volume of 250 µL. Fol-lowing the addition of stop buffer and Dowex-50 W (Na+) resin, the mixtures were transferred to spin cups, centrifuged and the formed L-[3H] citrulline contained in the flowthrough was quantified by scin-tillation counting27.

COX-2 activity assayFor measurements of COX-2 activity, the gastric mucosal cells from the control and various experimental treatments were settled by centrifu-gation, rinsed with phosphate-buff-ered saline and homogenized in 0.3 mL of cold sample 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 collected27. The COX-2 activity in 40 µL sample aliquots of the resulting supernatant was measured using COX activity assay kit (Cayman) in the absence and presence of COX-1 inhibition (SC-560), by monitoring the appearance of oxidized TMPD at 590 nm14.

As gastric mucosal responses to H. pylori are reflected in MAPK activa-tion9,14, in this study we investigated the relationship between H. pylori LPS-induced MAPK activation and the processes associated with EGFR transactivation. Our results dem-onstrate that the LPS-induced p38 activation along with MMPs are of critical significance to EGFR transac-tivation that leads to up-regulation in the activation of ERK signalling cas-cade, amplification in iNOS and COX-2 induction and consequently to the excessive increase in gastric mucosal PGE2 and NO generation.

Materials and methodsThe protocol of this study has be-en approved by the relevant et-hical committee related to our inst-itution in which it was performed. Animal care was in accordance with the institution guidelines.

Cell incubationThe gastric mucosal cells, collected from dissected rats with a blunt spat-ula, were suspended in five volumes of ice-cold Dulbecco’s modified (Gib-co) eagle’s minimal essential mediu-m (DMEM), supplemented with fung-izone (50 µg/mL), penicillin (50 U/mL), streptomycin (50 µg/mL) and 10% foetal calf serum. After gentle trituration with a syringe, the disper-sed cells were settled by centrifugati-on and resuspended in the medium at a concentration of 2 × 107 cells/mL27. Cell aliquots (1 mL) were then transferred to DMEM in culture dish-es and incubated under 95% O2–5% CO2 atmosphere at 37°C for up to 8 h in the presence of 0–100 ng/mL of H. pylori LPS6. H. Pylori used for LPS preparation was cultured from clini-cal isolates obtained from ATCC No. 43506. In the experiments evaluating the effect of EGF, EGFR kinase inhibi-tor, AG1478, and ascorbate (Sigma), p38 MAPK inhibitor, SB202190, ERK1/2 inhibitor, PD98059, Src inh-ibitor, PP2, Raf- 1 kinase inhibitor, iNOS inhibitor, 1400W, and a broad-

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a substantial suppression of the LPS-induced ERK1/2 phosphorylation, while the extent of p38 phosphoryla-tion remained unaffected (Figure 2). This attests to the involvement of EGFR transactivation in the media-tion of the LPS-induced ERK1/2 ac-tivation.

To gain further understanding of the mechanism of H. pylori LPS-induced EGFR transactivation, we next focused our attention on the pathways of EGFR activation. As EGFR activation in response to LPS

while the expression of COX-2 pro-tein was subject to suppression by the inhibitor of p38, SB202190. Fur-ther, following on recent literature evidence on the role of LPS in EGFR transactivation16, we assessed the MAPK requirement for H. pylori LPS-induced EGFR transactivation. For this, we examined the effect of EGFR kinase inhibitor, AG1478, on the LPS-induced phosphorylation of ERK1/2 and p38. The results of assays re-vealed that pretreatment of gastric mucosal cells with AG1478 leads to

NaF), containing 1 µg/mL leupeptin and 1 µg/mL pepstatin5. Following brief sonication, the lysates were cen-trifuged at 12,000×g for 10 min, and the supernatants were collected and normalized with respect to protein concentration using BCA protein as-say kit (Pierce). The samples, includ-ing 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 nitrocellulose 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 antibodies directed against ERK1/2, phospho-ERK1/2, p38, phospho-p38, COX-2 and iNOS (Calbiochem), EGFR, phospho-EGFR (Tyr1068), IKK-β (EMD Millipore) and phospho-IKK-β (Cell Signalling). The anti-β actin was from Sigma.

Data analysisAll experiments were carried out us-ing duplicate sampling, and the re-sults are expressed as means ± SD. Analysis of variance and nonpara-metric 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.

ResultsRecently, we reported that gastric mucosal inflammatory responses to H. pylori infection, characterized by the excessive NO and PGE2 genera-tion, are primarily linked to the LPS-induced activation of MAPK9. Indeed, as illustrated in Figure 1, incubation of rat gastric mucosal cells with H. pylori LPS leads to a significant in-duction in the level of iNOS and COX-2 proteins. Moreover, upon further examination, we found that the LPS-induced expression of iNOS protein exhibited susceptibility to inhibition by the ERK1/2 inhibitor, PD98059,

Figure 1: Effect of MAPK inhibitors on H. pylori LPS-induced changes in COX-2 and iNOS protein expression in gastric mucosal cells. The cells were treated with 30 µM of ERK1/2 inhibitor, PD98059 (PD), or 20 µM of p38 inhibitor, SB202190 (SB), and incubated for 8 h with the LPS at 100 ng/mL. Cell lysates were analysed by Western blotting for COX-2 and iNOS proteins (a) and their expression (b) were normalized to b actin. 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.

Figure 2: Effect of EGFR kinase inhibitor, AG1478, on H. pylori LPS-induced changes in gastric mucosal cell ERK1/2 and p38 phosphorylation. The cells, preincubated with 0 or 200 nM of AG1478 (AG), were incubated for 30 min with LPS at 100 ng/mL. Cell lysates were analysed by Western blotting for total and phosphorylated MAPK/ERK and p38 (a) and the relative densities of phosphorylated MAPK proteins (b) are expressed as a fold of control. Actin blot shows 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.

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has been reported to occur with the involvement of nonreceptor tyrosine kinase, Src as well as MMP-mediated release of EGFR ligand16,25, we em-ployed Src kinase-specific inhibitor, PP2, and a broad-spectrum MMP inhibitor, GM6001, in the assessment of the LPS-induced gastric mucosal EGFR phosphorylation. The results of Western blot analysis revealed that while Src inhibitor, PP2, had no effect on the extent of H. pylori LPS-induced EGFR phosphorylation, a significant decrease in the EGFR phosphoryla-tion was attained in the presence of MMP inhibitor, GM6001 (Figure 3). Moreover, the LPS-induced EGFR phosphorylation was susceptible to inhibition by p38 MAPK inhibi-tor, SB202190. However, neither the MMP nor p38 inhibitor affected the EGFR phosphorylation induced by EGF. Furthermore, similar require-ments for H. pylori LPS-induced gas-tric mucosal EGFR activation were also observed using EGRF protein tyrosine kinase activity assay (Figure 4). As shown in Figure 4, the LPS-in-duced EGFR activity was not affected by the inhibitors of Src and ERK1/2 kinases, but showed susceptibility to the inhibitors of p38 and MMP, the effect of which was additive. Col-lectively, these results indicate that EGFR transactivation by H. pylori LPS

Figure 3: Effect of Src, p38 and MMP inhibitors on H pylori LPS-induced EGFR transactivation in gastric mucosal cells. The cells, preincubated for 30 min with 30 µM PP2, 20 µM SB202190 (SB) or 100 µM GM6001 (GM), were treated with 100 ng/mL of LPS or 10 ng/mL EGFR and incubated for 10 min. Cell lysates were analysed by Western blotting for total and phosphorylated EGFR (a) and the relative density of pEGFR protein (b) is expressed a fold of control. Total EGFR was used as a loading control. The data represent the means ± SD of four experiments. *P < 0.05 compared with that of control. **P < 0.05 compared with that of LPS.

Figure 4: Effect of MAPK, Src and MMP inhibitors on H. pylori LPS-induced gastric mucosal EGFR protein tyrosine kinase activity. The gastric mucosal cells, preincubated for 30 min with 30 µM PP2, 30 µM PD98059 (PD), 20 µM SB202190 (SB) or 100 µM GM6001 (GM), were incubated for 15 min with LPS at 100 ng/mL. Values represent the means ± SD of four experiments. *P < 0.05 compared with that of control. **P < 0.05 compared with that of LPS.

occurs with the involvement of p38 MAPK and MMP and do not appear to require Src participation.

As gastric mucosal responses to H. pylori LPS associated with the activation of MAPK ERK through phosphorylation have been linked to the processes of IKK-β activation for the induction in NO and PGE2 production14, we next examined the role of EGFR transactivation in the LPS-induced ERK and IKK-β ac-tivation. Western blot analysis of the LPS-induced ERK1/2 phospho-rylation in the presence of EGFR in-hibitor, AG1478, revealed a partial decrease in ERK1/2 phosphoryla-tion, while the specific inhibitors of ERK1/2, PD98059 and RAF kinase, Raf-1 inhibitor, evoked nearly com-plete suppression in the ER1/2 phos-phorylation (Figure 5). Further, we found that the inhibitors of ERK1/2 and RAF kinase, PD9809 and Raf-1, exerted profound inhibitory effect on the LPS-induced activity of IKK-β, a key enzyme of NF-κB activation pathway (Figure 6). A significant decrease in the LPS-induced up-reg-ulation in gastric mucosal cell IKK-β activity was also attained in the pres-ence of EGFR inhibitor, AG1478, p38 MAPK inhibitor, SB202190, and MMP inhibitor, GM6001, while the inhibi-tor of Src, PP2, neither affected the

LPS-induced ERK1/2 phosphoryla-tion (Figure 5) nor the activity of IKK-β (Figure 6). These findings thus clearly point to the role of H. pylori LPS-induced EGFR transactivation in the amplification of ERK- mediated up-regulation in gastric mucosal IKK-β activity and subsequent induc-tion of proinflammatory iNOS and COX-2 enzymes.

Consequently, to provide ad-ditional support as to the role of EGFR transactivation in H. pylori LPS-induced up-regulation in NO and PGE2 generation, we assessed the activity of iNOS and COX-2 en-zymes in the presence of inhibitors of EGFR kinase, ERK and p38 MAPK. The results revealed that the LPS-elicited induction in the activity of iNOS and COX-2 enzymes was sus-ceptible to suppression by EGFR inhibitor, AG1478, as well as by the inhibitor of ERK1/2, PD98059 (Figure 7). The effect of p38 MAPK inhibitor, SB202190, was reflected mainly in the suppression COX-2 activity, whereas the inhibitor of Src, PP2, had no effect on either of the two enzymes. Moreover, prein-cubation with nitrosothiol reducing

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DiscussionInvasion of gastric mucosa by H. py-lori or stimulation of gastric mucosal cells with the bacterium LPS elicits a pattern of inflammatory responses characterized by the activation of MAPK cascade, induction in iNOS and COX-2 gene expression and the ex-cessive generation of NO and PGE2

1–

3,5,9,14. The data on the signalling events underlying the up-regulation in NO and PGE2 generation by LPS, moreover, point to the role of MAPK activation in the control of cognate transcription factors that exert con-trol over iNOS and COX-2 gene ex-pression8,10,12,30. Along these lines, we have recently provided evidence for the involvement of H. pylori LPS-elic-ited activation of p38 and ERK MAPK in the regulation of factors linked to the induction of gastric mucosal NO and PGE2 production9,14. As recent lit-erature evidence indicates that LPS-induced activation of MAPK cascade is also associated with EGFR transac-tivation15–17, in the present study we investigated the role of EGFR trans-activation in H. pylori LPS-stimulated induction in gastric mucosal iNOS and COX-2 expression.

Our data demonstrate that EGFR transactivation in gastric mucosal cells exposed to stimulation with H. pylori LPS is dependent on p38 MAPK and MMP activation and does not in-volve nonreceptor tyrosine kinase, Src, participation. We further show that the LPS-induced EGFR transac-tivation results in the amplification of ERK activity, up-regulation in ERK-mediated IKK-β activation and sub-sequent induction in the expression of iNOS and COX-2 enzymes. Indeed, we found that the LPS-induced EGFR phosphorylation as well as tyrosine kinase activity showed susceptibil-ity to the inhibitors of p38 MAPK and MMP, SB202190 and GM6001, but not that of Src inhibitor, PP2. Fur-thermore, neither MMP nor p38 in-hibitor affected the phosphorylation of EGFR induced by EGF, whereas blocking EGFR kinase activity with

together, these data suggest that H. pylori LPS-induced p38 MAPK activation along with MMPs plays the major role in EGFR transac-tivation that leads to amplifica-tion in the ERK signalling cascade, and consequently to up-regulation in gastric mucosal NO and PGE2 production.

agent, ascorbate, elicited a marked decrease in the LPS-induced COX-2 activity, but had no discernible effect on the extent of iNOS activa-tion. We also observed that the LPS-elicited induction in the activity iNOS and COX-2 enzymes was sus-ceptible to suppression by iNOS inhibitor, 1400W (Figure 7). Taken

Figure 6: Effect of Src, EGFR, RAF and MAPK inhibitors on H. pylori LPS-induced changes in gastric mucosal expression of IKK-b activity. The cells, preincubated with 30 µM PP2, 200 nM AG1478 (AG), 20 µM SB202190 (SB), 100 µM GM6001 (GM), 10 µM Raf-1 kinase inhibitor (Rf) or 30 µM PD98059 (PD), were incubated for 30 min with LPS at 100 ng/mL. Values represent the mean ± SD of four experiments. *P < 0.05 compared with that of control. **P < 0.05 compared with that of LPS.

Figure 7: Effect of ascorbate and EGFR, Src, ERK, p38 and iNOS inhibitors on H. pylori LPS-induced changes in the expression of iNOS and COX-2 activities in gastric mucosal cells. The cells, preincubated with 300 µM ascorbate (As), 200 nM AG1478 (AG), 30 µM PP2, 30 µM PD98059 (PD), 20 µM SB202190 (SB) or 40 µM 1400W (14W), were incubated for 8 h with LPS at 100 ng/mL. Values represent the mean ± SD of four experiments. *P < 0.05 compared with that of control. **P < 0.05 compared with that of LPS.

Figure 5: Effect of EGFR, Src, RAF and ERK inhibitors on H. pylori LPS-induced ERK1/2 phosphorylation in gastric mucosal cells. The cells, preincubated with 200 nM AG1478 (AG), 30 µM PP2, 10 µM Raf-1 kinase inhibitor (Rf) or 30 µM PD98059 (PD), were incubated for 30 min in the presence of 100 ng/mL of LPS. Cell lysates were analysed by Western blotting and for total and phosphorylated ERK1/2 (a), and the relative density of pER1/2 protein (b) is expressed as a fold of control. Total ERK was used as a loading control. The data represent the means ± SD of four experiments. *P < 0.05 compared with that of control. **P < 0.05 compared with that of LPS.

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linked to COX-2 activation through S-nitrosylation and the increase in PGE2 production30,33, and we have re-cently shown that suppression of H. pylori LPS-induced COX-2 S-nitros-ylation by iNOS inhibition results in the inhibition of PGE2 generation9. These data and the finding that the LPS-induced up-regulation in the ac-tivity of IKK-β was also susceptible to suppression by the inhibitors of ERK as well as EGFR transactivation, pro-vide strong indication as to the role of EGFR transactivation in the am-plification of ERK signalling cascade associated with up-regulation in gas-tric mucosal PGE2 and NO generation in response to H. pylori.

ConclusionThe data present in this report dem-onstrate the critical role of MAPK signalling cascade in the mediation of gastric mucosal inflammatory re-sponses to H. pylori LPS that lead to an up-regulation in gastric mucosal NO and PGE2 generation. The LPS-induced p38 MAPK activation along with MMPs plays a major role in EGFR transactivation that leads to amplification in ERK phosphoryla-tion, up-regulation in ERK-mediated IKK-β activation and increase in NF-κB nuclear translocation for the in-duction of iNOS expression and NO generation. The rise in iNOS-depend-ent NO, in turn, leads to COX-2 activa-tion through S-nitrosylation and ex-cessive PGE2 production (Figure 8). Thus, the extent of EGFR transactiva-tion elicited by H. pylori LPS may be of primary importance in defining the degree of gastric mucosal inflam-matory involvement that could also be of significance to the development of gastric carcinoma.

Abbreviations listCOX, cyclooxygenase; DMEM, Dul-becco’s modified Eagle’s minimal essential medium; EGFR, epidermal growth factor receptor; ERK, ex-tracellular signal-regulated kinase; HB-EGF, heparin-binding EGF-like

AG1478 leads to a substantial sup-pression in the phosphorylation of EK1/2, but had no effect on the ex-tent of the LPS-induced phospho-rylation of p38 MAPK. The fact that broad-spectrum MMP inhibitor, GM6001, affected the LPS-induced EGFR phosphorylation suggests the involvement of MMPs in the cleavage and release of EGFR ligand. While the nature of these MMPs was not inves-tigated, the demonstrated depend-ence of EGFR transactivation on the LPS-induced p38 MAPK activation points to MMP-2 or MMP-9, as the lit-erature data indicate that p38 MAPK plays an important role in the activa-tion of both these MMPs23,24,26,31.

Considering the demonstrated role of LPS-induced activation of Ras/Raf/MEK/MAPK pathway in the regulation of factors coupled to the induction of iNOS and COX-2 ex-pression, and the evidence as to the involvement of EGFR transactivation in ERK MAPK activation9,14,19,23,25, we further addressed the consequences of H. pylori LPS-induced EGFR trans-activation on the gastric mucosal ERK1/2 phosphorylation. A par-tial, but significant, reduction in the LPS-induced ERK1/2 phosphoryla-tion was attained with EGFR kinase inhibitor, AG1478, while RAF and ERK inhibitors, Raf-1 and PD98059, evoked nearly complete inhibition of the LPS-induced ERK1/2 phospho-rylation. These data thus suggest that EGFR transactivation associated with H. pylori infection could play a substantial role in the amplification of the LPS-induced gastric mucosal consequences of MAPK signalling cascade activation, including that of excessive NO and PGE2 genera-tion. Indeed, EGFR transactivation induced by LPS has been linked to the inflammatory up-regulation of COX-2 expression and PGE2 genera-tion in the intestine of infants af-fected by necrotizing colitis, as well as the exacerbation in inflammatory stimuli associated with biliary car-cinomas17,25. Moreover, ERK MAPK

has been implicated in a direct acti-vation of IKK-β complex responsible for NF-κB activation and iNOS gene induction14,32.

Accordingly, to ascertain further the involvement and the contribution of EGFR transactivation to H. pylori LPS-induced IKK-β complex activa-tion, we examined the gastric mu-cosal IKK-β activity in the presence of the inhibitors of EGFR kinase and ERK. We observed a profound inhibi-tory effect on the LPS-induced IKK-β activity in the presence of ERK1/2 in-hibitor, D98059, as well as RAF kinase inhibitor, Raf-1, while the inhibitors of EGFR, MMP and p38 MAPK pro-duced somewhat less pronounced suppression in the IKK-β activation. These data together with the results of our previous findings, linking the LPS-induced up-regulation in IKK-β activity to the enhancement in iNOS and COX-2 activation14, clearly sup-port the role of EGFR transactivation in the amplification of H. pylori LPS-induced proinflammatory processes associated with the excessive NO and PGE2 generation.

Indeed, assessment of the gastric mucosal activity of the iNOS and COX-2 enzymes revealed that the LPS-elicited induction in the activity of both enzymes was susceptible to suppression by EGFR kinase inhibi-tor, AG1478, as well as by ERK1/2 inhibitor, PD98059. The effect of p38 MAPK inhibitor, SB202190, was re-flected mainly in the suppression of COX-2 activity, while Src inhibitor, PP2, had no effect on either of the two enzymes. Furthermore, we found that nitrosothiol reducing agent, ascorbate, caused a marked decrease in the LPS-induced up-regulation in gastric mucosal COX-2 activity, but had no discernible effect on the ex-tent of iNOS activation. Moreover, induction in the extent of iNOS and COX-2 activation by the LPS was sus-ceptible to suppression by iNOS in-hibitor, 1400W. In this connection, it is pertinent to note that NO stimula-tion through iNOS induction has been

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pylori-associated gastritis may represent an increased risk factor to develop gastric carcinoma of the intestinal type. Int J Med Microbiol. 2003 Dec;293(6):403–12.2. Backert S, Neumann M. What a disorder:proinflammatory signalling pathways in-duced by Helicobacter pylori. Trends Mi-crobiol. 2010 Nov;18(11):479–86.3. Wroblewski LA, Peek RM, Wilson KT. Helicobacter pylori and gastric cancer: factors that modulate disease risk. Clin Microbiol Rev. 2010 Oct;23(4):713–39.4. Bauer B, Meyer TF. The human gastric pathogen Helicobacter pylori and its as-sociation with gastric cancer and ulcer disease. Ulcers.2011;2011:23.5. Slomiany BL, Slomiany A. Role of con-stitutive nitric oxide synthase in regula-tion of Helicobacter pylori-induced gas-tric mucosal cyclooxygenase-2 activation through S-nitrosylation: mechanism of ghrelin action. Open J Gastroenterol. 2011 Nov;1(2):13–22.6. Slomiany BL, Slomiany A. Role of ghre-lin-induced cSrc activation in modulation of gastric mucosal inflammatory respons-es to Helicobacter pylori. Inflammophar-macology. 2011 Aug;19(4):197–204.7. Joo M, Wright JG, Hu NN, Sadikot RT, Park Gy, Blackwell TS, et al. Yin yang 1 enhanc-es cyclooxygenase-2 gene expression in macrophages. Am J Physiol Lung Cell Mol Physiol. 2007 May;292(5):L1219–26.8. Lamon BD, Upmacis RK, Deeb RS, Ko-yuncu H, Haijar D. Inducible nitric ox-ide synthase gene deletion exaggerates MAPK-mediated cyclooxygenase-2 in-duction by inflammatory stimuli. Am J Physiol Heart Circ Physiol. 2010 Sep;299: H613–23.9. Slomiany BL, Slomiany A. Involvement of p38 MAPK-dependent activator pro-tein (AP-1) activation in modulation of gastric mucosal inflammatory responses to Helicobacter pylori by ghrelin. Inflam-mopharmacology 2013 Feb;21(1):67–78.10. Caivano M, Gorgoni B, Cohen P, Poli V. The induction of cyclooxygenase-2 mRNA in macrophages is biphasic and requires both CCAAT enhancer-binding protein β (C/EBPβ) and C/EBPδ tran-scription factors. J Biol Chem. 2001 Dec;276(52):48693–701.11. Grishin AV, Wang J, Potoka DA, Hack-am DJ, Uppermann JS, Boyle P, et al. Lipopolysaccharide induces cyclooxy-genase-2 in intestinal epithelium via a noncanonical p38 MAPK pathway. J Im-munol. 2006 Jan;176(1):580–8.

PGE2, prostaglandin; TLR, Toll-like receptor

References1. Reider G, Hofmann JA, Hatz RA, Stolte M, Enders, GA. Up-regulation of induc-ible nitric oxide synthase in Helicobacter

growth factor; IKK-β, inhibitory κB kinase-β; iNOS, inducible nitric oxide synthase; JNK, Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mi-togen-activated protein kinase; MMP, matrix metalloproteinase; NO, nitric oxide; NOS, nitric oxide synthase;

Figure 8: Schematic representation of the proposed mechanism involved in H. pylori LPS-elicited EGFR transactivation leading to the excessive gastric mucosal NO and PGE2 generation. Binding of the LPS to TLR4/MD4 initiates the activation of MAPKs and factors involved in the induction of COX-2 and iNOS expression. While JNK and p38 MAPKs promote the activation of factors (AP-1, CREB and C/EBP) implicated in the induction of COX-2 protein expression, activation of ERK MAPK leads to IKK-β-mediated activation of NF-κB for the induction in iNOS expression. Concomitantly, the LPS-activated p38 MAPK along with MMPs triggers the transactivation of EGFR that leads to the amplification in ERK phosphorylation, up-regulation in ERK-mediated IKK-β activation and the increase in NF-κB nuclear translocation for the enhanced induction in iNOS expression and NO generation. This leads to COX-2 activation through iNOS NO-dependent S-nitrosylation and the excessive PGE2 production. AP-1, activator protein-1; ATF-2, activating transcription factor-2; CREB, cAMP response element-binding protein; C/EBP, CCAAT/enhancer-binding protein; TLR4, Toll-like receptor 4.

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the epidermal growth factor receptor (EGFR) via ligand shedding, thereby enhancing colorectal cancer cell prolif-eration and migration. J Biol Chem 2012 Oct;287(42):35201–11.20. Schauwienold D, Sastre AP, Genzel N, Schaefer M, Reusch HP. The transac-tivated epidermal growth factor recep-tor recruits Pyk2 to regulate Src kinase activity. J Biol Chem. 2008 Oct;283(41): 27748–56.21. Bergin DA, Green CM, Sterchi EE, Kenna C, Geraghty P, Belaaouaj A, et al. Activation of epidermal growth fac-tor receptor (EGFR) by a novel metal-loprotease pathway. J Biol Chem. 2008 Nov;283(46):31736–44.22. Koff JL, Shao MX, Ueki IF, Nadel JA. Multiple TLRs activate EGFR via a sign-aling cascade to produce innate immune responses in airway epithelium. Am J Physiol Lung Cell Mol Physiol. 2008 Jun;294(6):L1068–75.23. Wetzke R, Bohmer FD. Transactiva-tion joins multiple tracks to the ERK/MAPK cascade. Nature Rev Mol Cell Biol. 2003 Aug;4(8):651–7.24. Spallarossa P, Altieri P, Garibaldi S, Ghigliotti G, Barisione C, Manca V, et al. Matrix metalloproteinase-2 and -9 are induced differently by doxorubicin in H9c2 cells: the role of MAP kinases and NAD(P)H oxidase. Cardiovasc Res. 2006 Feb;69(3):736–45 25. Finzi L, Shao MX, Paye F, Housset C, Nadel JA. Lipopolysaccharide initiates a positive feedback of epidermal growth factor receptor signaling by prosta-glandin E2 in human biliary carcinoma cells. J Immunol. 2009 Feb;182(4): 2269–76.26. Kumar B, Koul S, Petersen J, Khan-drika L, Hwa JS, Meacham RB, et al. p38 mitogen-activated protein kinase-driven MAPKAPK2 regulates invasion of blad-der cancer by modulation of MMP-2

12. Hou DX, MasuzakiS, Hashimoto F, Uto T, Tanigawa S, Fujii M, et al. Green tea proanthocyanidins inhibit cyclooxy-genase-2 expression in LPS-activated mouse macrophages: molecular mecha-nisms and structure-activity relation-ship. Arch Biochem Biophys. 2007 Apr; 460(1):67–74.13. Cho I, Kim SG. A novel mitogen-acti-vated protein kinase phosphatase-1 and glucocorticoid receptor (GR) interact-ing protein-1-dependent combinato-rial mechanism of gene transrepression by GR. Mol Endocrinol. 2009 Jan;23(1): 86–99.14. Slomiany BL, Slomiany A. Helicobac-ter pylori induction in gastric mucosal prostaglandin and nitric oxide genera-tion is dependent on MAPK/ERK-mediat-ed activation of IKK-β and cPLA2: Modu-latory effect of ghrelin. Open J Cell Biol. 2012;2:21–31.15. Fukata M, Chen A, Klepper A, Krishnareddy S, Vamadevan AS, Thomas LS, et al. Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: role in pro-liferation and apoptosis in the intestine. Gastroenterology. 2006 Sep;131(3): 862–77.16. Hsu D, Fukata M, Hernandez YG, So-tolongo JP, Goo T, Maki J, et al. Toll-like receptor 4 differentially regulates epider-mal growth factor-related growth factors in response to intestinal mucosal injury. Lab Invest. 2010 Sep;90(9):1295–305.17. McElroy SJ, Hobbs S, Kallen M, Tejera N, Rosen MJ, Grishin A, et al. Transac-tivation of EGFR by LPS induces COX-2 expression in enterocytes. PLoS One. 2012;7(5):e38373.18. Schlessinger J. Common and dis-tinct elements in cellular signaling via EGF and FGF receptors. Science. 2004 Nov;306:1506–7.19. Minder P, Bayha E, Becker-Pauly C, Sterchi EE. Meprinα transactivates

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in the amplification of Helicobacter pylori-elicited induction in ...(TLR-4), which then through a series of downstream effectors, causes the activation of transcriptional factors