JBC Origene Your Gene Company

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M007617200 on October 2, 2000

J. Biol. Chem., Vol. 275, Issue 50, 39779-39785, December 15, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/50/39779    most recent
M007617200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Foryst-Ludwig, A.
Right arrow Articles by Naumann, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Foryst-Ludwig, A.
Right arrow Articles by Naumann, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

p21-activated Kinase 1 Activates the Nuclear Factor kappa B (NF-kappa B)-inducing Kinase-Ikappa B Kinases NF-kappa B Pathway and Proinflammatory Cytokines in Helicobacter pylori Infection*

Anna Foryst-Ludwig and Michael NaumannDagger

From the Abteilung Molekulare Biologie, Max-Planck-Institut für Infektionsbiologie, Schumannstrasse 21/22, 10117 Berlin, Germany

Received for publication, August 21, 2000, and in revised form, September 29, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Helicobacter pylori, the causative agent of several human gastric diseases, induces activation of the immediate early response transcription factor nuclear factor kappa B (NF-kappa B), which subsequently triggers release of proinflammatory cytokines in colonized epithelial cells. Here we report that in H. pylori infection p21-activated kinase 1 (PAK1) activates NF-kappa B. Activated PAK1 associates with NF-kappa B-inducing kinase, which upon activation directs the activity of Ikappa B kinases to Ikappa Balpha . Our results indicate that in epithelial cells PAK1 participates in a unique pathway that links H. pylori-dependent effector molecules to the activation of NF-kappa B and the induction of the innate immune response.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Exposure of cells to various stimuli results in phosphorylation, ubiquitination, and subsequent degradation of Ikappa B molecules. The liberated nuclear factor kappa B (NF-kappa B)1 dimers are translocated to the nucleus, where they activate transcription of target genes (1). Key components of the intracellular signal transduction pathways regulating NF-kappa B activation are represented by NIK (2) and the Ikappa B kinases (IKKs) (3). IKKalpha and IKKbeta are the catalytic subunits of a protein kinase complex that phosphorylates Ikappa B molecules (4). IKKgamma (or NF-kappa B essential modulator (NEMO)) represents the regulatory subunit (5, 6).

We have studied the mechanism of NF-kappa B activation in response to human pathogenic Helicobacter pylori in epithelial cells where NF-kappa B, one of the main activators of the inflammatory response, triggers the induction of immune function including cytokine/chemokine production, growth control, and apoptosis (1). The epithelial cytokine/chemokine response is particularly important in the early stages of H. pylori-induced inflammation and is often followed by diseases like gastritis, peptic ulcer (7), gastric cancer (rarely), and low grade B-cell mucosa-associated lymphoid tissue gastric lymphoma (8). The major disease-associated, genetic difference in H. pylori strains is the presence or absence of a pathogenicity island (PAI) containing 31 genes, which code for proteins involved in a specialized type IV secretion machinery (9). H. pylori infection modulates the host cells by bacterial protein translocation. Several groups have recently shown for the first time that H. pylori translocates the CagA protein by a type IV secretion system. Tyrosine phosphorylation of CagA induces changes in the tyrosine phosphorylation state of proteins in the epithelial cell (10-14). Knockouts of certain PAI genes (CagA, CagF, and CagN), which are not necessary for the functional integrity of the type IV secretion apparatus, suppress or reduce the activation of NF-kappa B (15) and affect the secretion of cytokines/chemokines (16). Thus the integrity of the type IV secretion machinery is an absolute requirement for NF-kappa B activation in H. pylori infection. The identity of the effector molecule(s) of H. pylori and the mechanism of integration of the signals that induce NF-kappa B are not known so far. Therefore, we examined the mechanism of the H. pylori-induced activation of proinflammatory cytokine genes in the course of NF-kappa B activation in detail.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and H. pylori Infection-- Gastric epithelial cells (AGS) and 293 or HeLa cells were grown in RPMI 1640 containing 4 mM glutamine (Life Technologies, Inc.), 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (Life Technologies, Inc.) in a humidified 5% CO2 atmosphere. 16 h before infection, the medium was replaced by fresh RPMI 1640 medium supplemented with 0.1% fetal calf serum. The H. pylori P12 strain or the PAI strain (17) was cultured for 48-72 h on agar plates containing 10% horse serum in a microaerophilic atmosphere (generated by Campy Gen, Oxoid) at 37 °C. For the infection, the cell monolayer (50-70% confluence) was incubated with the bacteria to the multiplicities of infection (MOI) of 50 for different periods of time. Infection with H. pylori was routinely monitored by light microscopy.

Electrophoretic Mobility Shift Assay-- Nuclear extracts were prepared by using a nonionic detergent method as described previously (18). Detection of NF-kappa B was performed with a [alpha -32P]dATP-labeled Igkappa oligo probe containing the NF-kappa B recognition site. The DNA binding reactions were performed with 20 µl of binding buffer (2 µg of poly(dI-dC), 1 µg of bovine serum albumin, 5 mM dithiothreitol, 20 mM HEPES, pH 8.4, 60 mM KCl, and 10% glycerol) for 20 min at 30 °C. For competition experiments, the cold oligonucleotide probe was used, and supershift analysis was performed using antibodies against p65, p50, or c-Rel (18). The reaction products were analyzed via 5% polyacrylamide gel electrophoresis using 12.5 mM Tris, 12.5 mM boric acid, and 0.25 mM EDTA, pH 8.3. The gels were dried and exposed to Amersham TM film (Amersham Pharmacia Biotech) at -70 °C using an intensifying screen.

RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction-- Total RNA was isolated using Trizol reagent (Life Technologies, Inc.) as recommended by the manufacturer's instructions. Total RNA (1 µg) was reverse transcribed into single-stranded cDNA with Superscript IIRT (Life Technologies, Inc.) and oligo(dT) primers. Amplification of cytokine cDNAs and beta -actin cDNA as an internal control in each reaction was carried out by polymerase chain reaction with the primers as described previously (19). A subsaturating number of cycles allowed a semi-quantitative analysis within the infection kinetics. For inhibition of Ikappa Balpha degradation by the 26 S proteasome, the cells were preincubated for 60 min using 10 µM lactacystin (Affiniti) before the bacteria were added. Polymerase chain reaction products were visualized by ethidium bromide staining after agarose gel electrophoresis.

Transient Transfections and Reporter Assays-- Transactivating activity of NF-kappa B was analyzed in 293 or HeLa cells by cotransfection of a luciferase expression plasmid (400 ng) containing three repeats of the NF-kappa B human immunodeficiency virus-binding site and expression constructs using cationic liposomes (DAC-30, Eurogentec). 16 h after transfection cells were infected with H. pylori, treated with 10 ng ml-1 tumor necrosis factor alpha  (TNFalpha ) (Promega), or left untreated. Luciferase assays were performed 3-4 h after treatment as recommended by the manufacturer's instructions (Promega). The data presented are representative of more than three independent experiments. A pSV-beta -galactosidase vector (Promega) was used for normalization of transfection efficiency. The results were recorded on a Wallac 409 beta -counter (Berthold-Wallac) and given as fold induction or as percent induction compared with the control.

Immunoprecipitation and Protein Kinase Assays-- From the radioimmune precipitation buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 2.5% glycerol, 1 mM EGTA, 50 mM NaF, 1 mM Na3VO4, 10 mM Na4P2O7, 100 µM phenylmethylsulfonyl fluoride, 10 µM pepstatin A, and 4 µM aprotinin) lysed cell immunoprecipitations were performed with anti-alpha -PAK (sc-881, Santa Cruz Biotechnology), anti-IKKalpha (Pharmingen), anti-IKKbeta (BIOSOURCE), anti-Ha (sc-805, Santa Cruz Biotechnology), anti-Vsv (Roche Molecular Biochemicals), anti-Myc (Pharmingen), or anti-Flag (Sigma) antibodies. The immunoprecipitated and co-immunoprecipitated proteins were separated via SDS-PAGE and blotted as described previously (18). Immunodetection was achieved with antibodies as indicated (anti-alpha -PAK1, sc-882 (Santa Cruz Biotechnology); anti-Ikappa Balpha , sc-847 (Santa Cruz Biotechnology); anti-NEMO (BIOSOURCE), etc.). For in vitro kinase reactions, immunocomplexes were recovered, washed, and incubated with the kinase buffer (100 mM KCl, 0.1 mM CaCl2, 6 mM MgCl2, 30 mM Tris, pH 7.5, 0.1 mM Na3VO4, 1 mM dithiothreitol, 10 µCi of [gamma -32P]ATP) using 2.5 µg of myelin basic protein (Upstate Biotechnology) and 0.5 µg of Ikappa Balpha (18) as substrates for PAK1 and IKKs, respectively. The samples were separated via SDS-PAGE and dried, and phosphorylation was visualized by autoradiography. Equal amounts of the total lysates were analyzed in an immunoblot to indicate equivalent protein amounts in all lanes. In vitro translation was performed using a TNT kit (Promega), and coimmunoprecipitation was performed with translation reactions diluted in phosphate-buffered saline.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NF-kappa B Activation in H. pylori Infection Involves IKK Activity-- AGS cells (neuroendocrine differentiated gastric carcinoma cells) infected with H. pylori or treated with TNFalpha for various periods of times, fractionated into cytoplasmic and nuclear components, and subjected to electrophoretic mobility shift assay show an equivalent increase of NF-kappa B DNA binding activity within 90 and 10 min, respectively (Fig. 1a, lanes 1-9). The H. pylori-induced binding activity was strongly reduced and partially super-shifted when anti-p50 or anti-p65 antibodies were used, whereas an anti-c-Rel antiserum or preimmune serum did not show a reduction (Fig. 1a, lanes 10-13). The specificity of the DNA binding activity was examined by adding increasing amounts of the non-labeled double-stranded Igkappa oligonucleotide for competition (Fig. 1a, lanes 14-16). In contrast to the wild type H. pylori strain, an isogenic mutant strain (PAI, no expression of the type IV secretion machinery) did not show NF-kappa B activation (Fig. 1a, lanes 17-19). This experiment excludes a role for lipopolysaccharide in H. pylori-induced NF-kappa B activation in epithelial cells. To determine whether NF-kappa B activation is sustained at later stages of the infection, we studied H. pylori infection up to 6 h after infection. Maximal DNA binding of NF-kappa B was observed between 90 and 180 min after infection and was not detectable later than 240 min after infection (Fig. 1a, lanes 20-22). De novo protein synthesis was not required for H. pylori-induced NF-kappa B activation (data not shown). Furthermore, H. pylori infection or TNFalpha stimulation led to a rapid loss of Ikappa Balpha as analyzed in an immunoblot (Fig. 1b). To assess changes in cytokine gene expression, AGS cells were challenged to infection with H. pylori. Cytokine mRNA expression was determined at different time points after infection by reverse transcriptase-polymerase chain reaction of total RNA prepared from infected cells. The cytokine mRNA levels were compared with the constitutive beta -actin mRNA in the same polymerase chain reactions. Infection with H. pylori led to an increased synthesis of granulocyte-macrophage colony-stimulating factor, TNFalpha , and interleukin-8 as soon as 45 min after infection (Fig. 1c, lanes 1-4). Treatment of the cells with the proteasome inhibitor lactacystin prior to H. pylori infection suppressed the cytokine mRNA expression (Fig. 1c, lanes 5-8). The correlation of NF-kappa B activation and Ikappa Balpha degradation, which is blocked by a proteasome inhibitor, demonstrates that cytokine/chemokine genes are indeed subject to a coordinate cellular regulation in response to H. pylori infection.



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 1.   Induction of cytokine genes and activation of NF-kappa B in H. pylori-colonized AGS cells. a, NF-kappa B activation. Electrophoretic mobility shift assay of AGS cells colonized by H. pylori wild type or PAI strains (MOI 50) (lanes 1-6 and 17-22) or of TNFalpha -treated cells (10 ng ml-1) (lanes 7-9) at the indicated periods of time; antibody supershifting of the NF-kappa B DNA complex (lanes 10-13) or cold oligonucleotide competition (5, 10, or 50 ng) (lanes 14-16). p.s., preimmune serum; ns, nonspecific complex. b, Ikappa Balpha degradation. Immunoblot of H. pylori-infected AGS cells using anti-Ikappa Balpha antibody (sc-847, Santa Cruz Biotechnology). c, induction of cytokine mRNAs. Duplex reverse transcriptase-polymerase chain reaction of the cytokine genes granulocyte-macrophage colony-stimulating factor (upper panel), interleukin-8 (middle panel), and TNFalpha (lower panel) in response to H. pylori in the absence (lanes 1-4) or presence (lanes 5-8) of 10 µM lactacystin. Hp, H. pylori.

To address whether H. pylori-induced NF-kappa B activation and Ikappa Balpha degradation involves IKK activity, we tested the ability of H. pylori to induce phosphorylation of endogenous IKKs. AGS cells were infected for the indicated periods of time. IKKalpha or IKKbeta (Fig. 2, a and b) were immunoprecipitated from cell extracts with appropriate antibodies and analyzed subsequently in an in vitro kinase assay using Ikappa Balpha as a substrate. H. pylori infection as well as TNFalpha treatment of cells resulted in increased IKKalpha (Fig. 2a, lanes 1-8, upper panel) and IKKbeta (Fig. 2b, lanes 1-8, upper panel) kinase activity on the substrate Ikappa Balpha . Compared with H. pylori-infected cells, TNFalpha -treated cells induced IKKalpha and IKKbeta faster in the analyzed time course as expected. Immunoblot analysis confirmed the presence of similar quantities of IKK proteins in each of the extracts used for immunoprecipitation (Fig. 2, a and b, lower panels).



View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2.   H. pylori-induced NF-kappa B activation involves the IKK complex. a and b, H. pylori induces IKKalpha and IKKbeta . AGS cells were infected with H. pylori (MOI 50) or treated with TNFalpha for the indicated time periods. Endogenous IKKalpha (a) or IKKbeta (b) was immunoprecipitated from lysed cells, and their kinase activity was determined using Ikappa Balpha as substrate. Autoradiographs from SDS-PAGE are shown. IP, immunoprecipitation; IB, immunoblot. c and d, kinase-inactive IKKbeta blocks the H. pylori-induced transcriptional activity of NF-kappa B. Relative luciferase activity in 293 cells induced by H. pylori (MOI 50) or TNFalpha (10 ng ml-1) (c) or co-transfected with Igkappa reporter plasmid and kinase-inactive IKKbeta (K44A) or MKK4(K116R) (d) and expression vectors, as indicated. The results are representative of at least three independent experiments. Data are expressed as fold-activation compared with non-treated cells or as percent induction. Hp, H. pylori.

To analyze the ability of H. pylori to induce NF-kappa B transactivation activity, we tested the effects of activation of H. pylori on the expression of an NF-kappa B-dependent reporter gene in transiently transfected 293 cells. H. pylori infection (MOI 50) or TNFalpha treatment of cells increased transcription of the reporter gene (Fig. 2c). When we tested the effect of a kinase-inactive IKKbeta (K44A) construct on the expression of an NF-kappa B-dependent reporter gene in transiently transfected and H. pylori-infected or TNFalpha -treated 293 cells, we observed suppression of NF-kappa B activation. This indicates that H. pylori-induced NF-kappa B activation involves IKKbeta (Fig. 2d). For a control, we analyzed the effect of kinase-inactive MAP kinase kinase (MKK4(K116R)) on the H. pylori-induced NF-kappa B activation, which was not affected.

PAK1 Activates NIK and the IKK Complex-- Several kinases (e.g. NIK, mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK) kinase kinase 1 (MEKK1), IKK-i, TANK-binding kinase 1/NF-kappa B-activating kinase, mixed-lineage kinase 3) have been shown to be signaling intermediates that act as direct activators of the IKK complex (2, 20-24). The cellular selection of the kinases might be dependent on cell type specificity and distinct extracellular stimuli. To study the H. pylori-induced upstream kinase involved in activation of the IKK complex, we analyzed the role of NIK and the MEKK1 in H. pylori-induced expression of an NF-kappa B-dependent reporter gene in transiently transfected 293 cells. H. pylori- or TNFalpha -stimulated NF-kappa B-dependent reporter gene activity was suppressed when kinase-inactive mutants of NIK (NIK(624-947); NIK(K429A,K430A)) were expressed (Fig. 3a). In contrast to kinase-inactive NIK, the kinase-inactive MEKK1(K432M) did not significantly block H. pylori-induced NF-kappa B activation (Fig. 3a), whereas phorbol 12-myristate 13-acetate-induced NF-kappa B activation was affected by MEKK1(K432M) (data not shown). These results suggest that H. pylori-induced NF-kappa B activation involves NIK but not MEKK1 in this kinase cascade and exclude a functional cooperation between those kinases in the activation of IKK activity. Compelling evidence that NIK is involved in the activation of NF-kappa B has been shown in response to CD40 and CD3/CD28 induction (25, 26).



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 3.   NIK and PAK1 direct H. pylori-induced activation of NF-kappa B. a, kinase-inactive NIK inhibits H. pylori-induced transcriptional activity of NF-kappa B. Relative luciferase activity in 293 cells induced by H. pylori (MOI 50) or TNFalpha (10 ng ml-1) or co-transfected with Igkappa reporter plasmid and kinase-inactive NIK ((NIK(K429A,K430A) and NIK(624-947)) or MEKK1(K432M) and expression vectors, as indicated. The results are representative of at least three independent experiments. Data are expressed as fold-activation compared with non-treated cells or as percent induction. b, H. pylori infection induces PAK1. Immunocomplex kinase activity of PAK1 immunoprecipitated with an anti-alpha -PAK antibody from H. pylori-infected (MOI 50) (lanes 1-4) or TNFalpha -stimulated (10 ng ml-1) (lanes 5-7) AGS cells at the indicated periods of time. PAK1 activity was determined by phosphorylation of myelin basic protein as a substrate (upper panel). The immunoblot of endogenous PAK1 shows similar protein amounts in all lanes (lower panel). MBP, myelin basic protein; IP, immunoprecipitation; IB, immunoblot. c, PAK1 is involved in H. pylori-induced transcriptional activity of NF-kappa B. 293 cells were co-transfected with an Igkappa reporter plasmid and expression vectors encoding different kinase-inactive PAK1s (as indicated). The relative luciferase activity was determined in H. pylori-infected (MOI 50) or TNFalpha -treated (10 ng ml-1) cells. Data are expressed as percent induction. d, PAK1 is upstream of NIK. HeLa cells were co-transfected with an Igkappa reporter plasmid and expression vectors encoding constitutive active PAK1(L107F) (500 ng) and kinase-inactive NIK(K429A,K430A) or NIK wild type (100 ng) and different kinase-inactive PAK1 constructs (PAK1(K299R) and PAK1(H83L,H86L,K299R)). The results are representative of at least three independent experiments. Data are expressed as percent induction. Hp, H. pylori.

Our experimental evidence that the MAPK kinase kinase NIK represents a crucial factor in H. pylori-directed activation of the IKK complex led us to search for putative upstream regulators of NIK. Colinear amino acid sequence alignment of the kinase domain in NIK revealed homology to MEKK1 and Ste11 apart from other kinases, and Ste11 represents a downstream kinase of Ste20, the yeast homologue of mammalian PAK1 (27). Therefore, we examined whether PAK1, which is a stress response kinase of the MAPK kinase kinase kinase level, represents an upstream kinase for NIK. In AGS cells the activity of PAK1 was induced severalfold in response to pathophysiological stress raised by H. pylori infection. The kinase activity of immunoprecipitated PAK1 was measured with myelin basic protein as a substrate (Fig. 3b, lanes 1-4, upper panel). TNFalpha weakly induced PAK1 (Fig. 3b, lanes 5-7, upper panel). Immunoblot analysis confirmed the presence of similar quantities of PAK1 in each of the extracts used for immunoprecipitation (Fig. 3b, lower panel).

To study the mechanism of NF-kappa B activation, we analyzed whether PAK1 affects the H. pylori-induced NF-kappa B-dependent transcriptional activity. Kinase-inactive PAK1(K299R) blocked the expression of an NF-kappa B-dependent reporter gene in transiently transfected and H. pylori-infected 293 cells in a dose-dependent manner (Fig. 3c). In contrast to H. pylori infection, kinase-inactive PAK1(K299R) only slightly affected the NF-kappa B transactivation activity in TNFalpha -treated cells (Fig. 3c). To exclude possible effects on the putative upstream components of PAK1 due to titration of Rho-GTPases, we performed experiments with a PAK1(K299R) construct mutated in histidine 83 and 86, which prevents the binding of Rho-GTPases. This construct clearly blocked H. pylori-induced NF-kappa B activation. Furthermore, we used a PAK1 construct containing the residues 83-149. PAK1(83-149) has been demonstrated to inhibit the autophosphorylation of PAK1 by blocking a critical phosphoacceptor site that is required for its kinase activity and does not bind to Rac1 (28). Overexpression of this construct blocked H. pylori-induced activation of NF-kappa B in a dose-dependent manner (Fig. 3c). For a control, we transiently transfected a PAK1(83-149) construct with the leucine 107 to phenylalanine mutation (PAK1(83-149,L107F)), which inactivates the autoinhibitory domain of PAK1. This construct did not block H. pylori-induced NF-kappa B activation. To determine the signaling pathways that couple PAK1 to NF-kappa B activation, we tested whether kinase-inactive NIK(K429A,K430A) could block signaling from constitutive active PAK1(L107F) in transiently transfected HeLa cells. The inactive NIK mutant NIK(K429A,K430A) blocked the activation of NF-kappa B-dependent gene expression by PAK1(L107F), whereas overexpression of inactive PAK1 (PAK1(K299R) and PAK1(H83L,H86L,K299R)) had little effect on NIK-induced activation of NF-kappa B (Fig. 3d). Our results raise the possibility that PAK1 may function as an activator of NIK to mediate NF-kappa B activation in H. pylori infection.

To identify NIK as a potential downstream component of PAK1 in H. pylori infection, we studied the interaction of these kinases. AGS or HeLa cells were transfected with Ha-tagged PAK1 constructs or Flag-tagged NIK constructs. The kinases were then immunoprecipitated. The immunoprecipitates were subjected to SDS-PAGE and analyzed in an immunoblot with the indicated antibodies. We observed interaction of Ha-PAK1(K299R) with Flag-NIK (Fig. 4a, lane 1), high affinity interaction between Ha-PAK1(K299R) and Flag-NIK(K429A,K430A) (lane 2), and interaction of Ha-PAK1 with Flag-NIK(K429A,K430A) (lane 3). We also performed these experiments with constitutive active Ha-PAK1(L107F), which resulted in a detectable interaction with Flag-NIK(K429A,K430A) (lane 4). These results indicate that kinase-active PAK1 could target the MAPK kinase kinase NIK. In contrast to the PAK1/NIK interaction, we observed no direct interaction between PAK1 and the IKK complex components IKKalpha , IKKbeta , or NEMO/IKKgamma (Fig. 4, b and c). Appropriate controls show that NIK interacted with IKKalpha (Fig. 4b) and that IKKbeta interacted with NEMO/IKKgamma (Fig. 4c). Similar to the kinase-inactive PAK1(K299R), the constitutively active PAK1(L107F) did not interact with an IKK subunit (data not shown).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 4.   PAK1 binds NIK but not IKKs. a-c, PAK1 binds NIK. HeLa cells were transiently transfected, and the tagged proteins (as indicated) were immunoprecipitated from cell lysates. The immunoprecipitates were subjected to SDS-PAGE and analyzed in an immunoblot with the indicated antibodies. d, H. pylori induces the interaction between PAK1 and NIK. To analyze endogenous PAK1/NIK interaction, we transiently transfected in HeLa cells small amounts of kinase-inactive Flag-NIK(K429A,K430A) and performed immunoprecipitations from cell lysates of H. pylori-infected (MOI 50) (lanes 1-4, upper panel) or TNFalpha -treated cells (lanes 5 and 6, upper panel) with an anti-alpha -PAK1 antibody. Immunoblot analysis confirmed the presence of similar quantities of Flag-NIK and endogenous PAK1 in each of the extracts used for immunoprecipitation (middle and lower panels). e, the interaction between PAK1 and NIK is direct. Protein-protein interaction was carried out in an in vitro analysis using 35S-labeled proteins translated in wheat germ extracts (Promega). After translation of Flag-NIK (lane 2) and HaPAK1 (lane 3) and co-translation of both molecules (lane 1), we performed a co-immunoprecipitation with an anti-Flag antibody (lanes 4-6). The position of proteins are indicated. IP, immunoprecipitation; IB, immunoblot.

To analyze endogenous PAK1/NIK interaction, we tried to derive HeLa cells stably expressing an epitope-tagged version of NIK. Unfortunately, cells which had integrated the NIK wild type cDNA did not survive. To circumvent this problem we transiently transfected small amounts of kinase-inactive Flag-NIK(K429A,K430A) in a titration experiment and determined an amount that still allowed interaction with activated PAK1 induced in H. pylori infection. Interestingly, H. pylori infection caused an inducible interaction of the endogenous PAK1 and the kinase-inactive Flag-NIK(K429A,K430A) (Fig. 4d, lanes 2-4, upper panel), whereas the non-stimulated or TNFalpha -treated cells exhibited no PAK1/NIK interaction (lanes 1, 5, and 6, upper panel). Immunoblot analysis confirmed the presence of similar quantities of Flag-NIK and endogenous PAK1 in each of the extracts used for immunoprecipitation (Fig. 4d, middle and lower panels). To study whether this interaction is direct, we carried out an in vitro analysis using 35S-labeled proteins translated in wheat germ extracts. After cotranslation of Flag-NIK and HaPAK1 (Fig. 4e, lane 1) followed by anti-Flag immunoprecipitation (lane 4), we detected PAK1 in the immunoprecipitate. The converse experiment of immunoprecipitation with an anti-Ha antibody allowed the detection of Flag-NIK in the immunoprecipitate (data not shown).

Our results raised the possibility that PAK1 could function as the kinase that activates NIK in H. pylori infection. The analysis of H. pylori-infected cells, which were transfected with kinase-inactive NIK(K429A,K430A), resulted in the occurrence of an additional NIK form with a slightly decreased mobility in SDS-PAGE as detected in an immunoblot (Fig. 5a, lane 2). When NIK(K429A,K430A) was expressed without H. pylori infection, this slower migrating form of NIK was weakly visible (Fig. 5a, lane 1). Cells cotransfected with constitutive active PAK1(L107F) caused an accumulation of the more slowly migrating band of NIK(K429A,K430A) (Fig. 5b, lane 4), whereas cotransfection with kinase-inactive PAK1(K299R) suppressed this slower band (lane 3). The upshifted band of NIK in H. pylori-infected cells was eliminated by lambda  phosphatase treatment (Fig. 5a, lane 5), suggesting that it represented phosphorylated NIK. These data suggest that, once activated, PAK1 binds and phosphorylates NIK.



View larger version (29K):
[in this window]
[in a new window]
  		Fig. 5.   PAK1 phosphorylates NIK. a, effect of PAK1 on phosphorylation of NIK. HeLa cells were transfected with Flag-NIK(K429A,K430A) (lane 1) and infected with H. pylori (lane 2) or additionally co-transfected with kinase-inactive HaPAK1(K299R) (lane 3) or co-transfected with constitutive active PAK1(L107F) (lane 4). The identity of the phosphoshift was determined by lambda  phosphatase treatment of H. pylori-infected cells (lane 5). The phosphoshift is indicated with an arrow. lambda PP, lambda  phosphatase. b, effect of PAK1 on phosphorylation of IKKalpha . HeLa cells were transfected with IKKalpha and stimulated with TNFalpha (lane 2), co-transfected with PAK1(K299R) (lane 3); cells were also infected with H. pylori (lane 4) or additionally co-transfected with PAK1(K299R) (lane 5) or NIK(K429A,K430A) (lane 6). The IKKalpha immunoprecipitations were performed using an anti-Vsv antibody, and the immunocomplex kinase reactions were analyzed by SDS-PAGE and autoradiography. The phosphorylated IKKalpha (IKKalpha -P) is indicated (top panel). The other panels show the immunoblot analysis, which confirms the presence of similar quantities of Vsv-IKKalpha , Flag-NIK, and HaPAK1 in each of the extracts used for immunoprecipitation. IP, immunoprecipitation; IB, immunoblot.

To show that PAK1 is critical for H. pylori-induced activation of the IKK complex, IKKalpha was cotransfected with kinase-inactive PAK1(K299R), and the cells were either infected with H. pylori or stimulated with TNFalpha . Kinase assays of immunoprecipitated IKKalpha were performed, and the effect of kinase-inactive PAK1 on IKKalpha phosphorylation was analyzed (Fig. 5b). Kinase-inactive PAK1(K299R) (Fig. 5b, lane 5) as well as NIK(K429A,K430A) (lane 6) could efficiently block the H. pylori-induced IKKalpha phosphorylation (lane 4), whereas TNFalpha -induced IKKalpha phosphorylation (lane 2) was not blocked by PAK1(K299R) (lane 3). These findings strongly suggest that PAK1 and NIK represent crucial components in H. pylori-induced NF-kappa B activation. Thus, like Cot/TPL2, TAK1(TAB1/TAB2), Raf, and PKCtheta (26, 29-34), the activation of PAK1 results in the activation of NF-kappa B by certain stimuli, but distinct from pathways used by TNFalpha . The activation of the IKK complex by these kinases is indirect and involves other kinases or adaptor molecules, and not all signals proceed through IKKalpha . It has been shown that PKCtheta -induced signaling targets IKKbeta (35) and that the adaptor protein receptor-interacting protein targets NEMO/IKKgamma to recruit the IKK complex to the TNF receptor (36), which could involve TRAF2 (37).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In summary, our results identify PAK1 as an integral component of the H. pylori-induced activation of NF-kappa B in epithelial cells. Activated PAK1 phosphorylates NIK and directs the activity of IKKs to Ikappa Balpha . This signaling causes NF-kappa B activation and induction of proinflammatory cytokines in H. pylori-infected gastric epithelial cells (Fig. 6). Because PAK1 does not play an important role in NF-kappa B activation by TNFalpha or phorbol 12-myristate 13-acetate, this study corroborates the concept that different NF-kappa B-activating stimuli use different signaling components.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 6.   Model for the role of PAK1 in the activation of the NIK-IKK NF-kappa B pathway and the induction of the innate immune response in H. pylori infection. See "Results".

Because the integrity of the type IV secretion machinery is an absolute requirement for NF-kappa B activation in H. pylori infection (38), it is reasonable to speculate that the component(s) that induce(s) this transcription factor must either be injected into the eucaryotic target cell or activate(s) contact-dependent eucaryotic cell-surface receptors. This hypothesis is supported by the notion that only those PAI factors (CagA, CagF, and CagN) that are not necessary for the functional integrity of the type IV secretion apparatus do not induce activation of NF-kappa B (15). Thus PAK1 functions in gastric epithelial cells in a unique pathway that links H. pylori-dependent effector molecules to the activation of NF-kappa B and the induction of the innate immune response. The central role of NF-kappa B in the innate immune response suggests that many pathogenic microorganisms have evolved mechanisms to interfere with the function of NF-kappa B or modulate the NF-kappa B signal transduction. In contrast to H. pylori, the microbial pathogen Yersinia pseudotuberculosis inhibits the activation of NF-kappa B by active translocation of the virulence factor yersinia outer protein J via a type III secretion system. The yersinia outer protein J protein was shown to bind to IKKbeta and the superfamily of mitogen-activated protein mitogen-activated protein kinase kinases and inhibits phosphorylation and subsequent activation of these kinases (39). Thus, by blocking IKKbeta and MKKs Yersinia inhibits the release of cytokines and other immunomodulatory factors during innate immune response. The identity of the effector molecule(s) of H. pylori that induce(s) NF-kappa B is not known so far. We speculate that PAK1 becomes targeted directly or indirectly by H. pylori factor(s). The identification of the H. pylori factor(s) in this process of NF-kappa B activation will unravel the mechanism of the H. pylori-induced signaling in the future. Interestingly, the viral protein negative regulatory factor from human simian immunodeficiency virus targets PAK1 and induces nuclear responses. The negative regulatory factor-induced mechanism of PAK1 activation involves the simultaneous interaction of negative regulatory factor with the SH3 domain of Vav, which induces its guanine nucleotide exchange factor activity for Cdc42 and Rac1. The activation of Rac1 and Cdc42 leads to their subsequent dissociation from Vav and strongly increases their affinity for PAK1 (40).

Based on their structure the PAK kinases in mammals can be divided into two subfamilies: the PAK subfamily, containing an NH2-terminal catalytic p21-binding domain (also known as cdc42/Rac1-interactive binding domain) and a COOH-terminal kinase domain, and the germinal center kinase-like subfamily, which contains an NH2-terminal catalytic domain and lacks the p21-binding domain. In the first description of PAK it was shown that the serine/threonine protein kinase activity of PAK could be stimulated by the binding of activated GTP-bound Rac1 and Cdc42. More recently, a variety of studies have suggested that PAKs can participate in a broad range of cellular events that include cytoskeletal responses as well as certain signaling events (41). For example, PAKs can catalyze the phosphorylation of the heavy chain in myosin 1 (42), in p47-phox (43), and in LIM kinase (44). In our study we have shown for the first time that PAK1 interacts with and phosphorylates NIK, which represents a specific activating mechanism in NF-kappa B regulation in H. pylori-infected epithelial cells. The recent observation that PAK1-mediated NF-kappa B activation does not involve activation of the IKKs could be explained by a different experimental approach (45). In our future work we plan to analyze the whole process of H. pylori-induced NF-kappa B activation including the identification of the microbial factors. Regarding the phosphorylation of NIK by PAK1, further experiments will determine the phosphorylation sites. NIK putatively contains a consensus site for phosphorylation by PAK (46). Therefore, NIK phosphorylation could take place in the NH2-terminal region, possibly activating NIK to autophosphorylate and stimulate catalytic activity, as proposed for the activation of NIK by TAK1 (29). Because PAK1 is known to activate the JNK pathway in H. pylori infection (47), this finding contributes to our understanding of how a given stimulus can simultaneously activate JNK as well as NF-kappa B. Other kinases could also act as dual activators of JNK and NF-kappa B, e.g. MEKK1 or mixed-lineage kinase 3 activate JNK via phosphorylation of MKK4 (48, 49) and trigger NF-kappa B activation by direct phosphorylation of IKKs (24, 35). Furthermore, dual activation of JNK and NF-kappa B activities has been shown by ectopic expression of plenty of SH3s, a Rac1-regulated protein, which consists of four SH3 domains (50).

Gastric inflammation is a hallmark of H. pylori infection, and our work contributes to the elucidation of the steps involved in H. pylori-induced NF-kappa B signal transduction and activation of an innate host immune response. The understanding of the host cell response mechanism may reveal potential targets for drug intervention in the case of pathological activation of this transcription factor in inflammatory diseases.


    ACKNOWLEDGEMENTS

We are grateful to Jonathan Chernoff, David Wallach, Edward Manser, Alain Israel, and Claus Scheidereit for cDNA constructs. We would like to thank Birte Wolff for critical reading of the manuscript and Björn Wieland for excellent technical assistance.


    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant Na 292/5-1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Abteilung Molekulare Biologie, Max-Planck-Institut für Infektionsbiologie, Schumannstr. 21/22, 10117 Berlin, Germany. Tel.: 49-30-28460-410; Fax: 49-30-28460-401; E-mail: naumann@mpiib-berlin.mpg.de.

Published, JBC Papers in Press, October 2, 2000, DOI 10.1074/jbc.M007617200


    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; NIK, NF-kappa B-inducing kinase; IKK, Ikappa B kinase; NEMO, NF-kappa B essential modulator; PAI, pathogenicity island; MOI, multiplicities of infection; TNF, tumor necrosis factor; PAK, p21-activated kinase; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; MEKK1, mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK) kinase kinase 1; JNK, c-Jun NH2-terminal kinase.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Hatada, E. N., Krappmann, D., and Scheidereit, C. (2000) Curr. Opin. Immunol. 12, 52-58
2. Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature 385, 540-544
3. Delhase, M., Hayakawa, M., Chen, Y., and Karin, M. (1999) Science 284, 309-313
4. Zandi, E., and Karin, M. (1999) Mol. Cell. Biol. 19, 4547-4551
5. Yamaoka, S., Courtois, G., Bessia, C., Whiteside, S. T., Weil, R., Agou, F., Kirk, H. E., Kay, R. J., and Israel, A. (1998) Cell 93, 1231-1240
6. Rothwarf, D., Zandi, E., Natoli, G., and Karin, M. (1998) Nature 395, 297-300
7. Blaser, M. J. (1987) Gastroenterology 93, 371-383
8. Parsonnet, J., Hansen, S., Rodriguez, L., Gelb, A. B., Warnke, R. A., Jellum, E., Orentreich, N., Vogelman, J. H., and Friedman, G. D. (1994) N. Engl. J. Med. 330, 1267-1271
9. Covacci, A., Telford, J. L., Del Giudice, G., Parsonnet, J., and Rappuoli, R. (1999) Science 284, 1328-1333
10. Segal, E. D., Cha, J., Lo, J., Falkow, S., and Tompkins, L. S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 14559-14564
11. Stein, M., Rappuoli, R., and Covacci, A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1263-1268
12. Asahi, M., Azuma, T., Ito, S., Ito, Y., Suto, H., Nagai, Y., Tsubokawa, M., Tohyama, Y., Maeda, S., Omata, M., Suzuki, T., and Sasakawa, C. (2000) J. Exp. Med. 191, 593-602
13. Odenbreit, S., Püls, J., Sedlmaier, B., Gerland, E., Fischer, W., and Haas, R. (2000) Science 287, 1497-1500
14. Backert, S., Ziska, E., Brinkmann, V., Zimny-Arndt, U., Fauconnier, A., Jungblut, P., Naumann, M., and Meyer, T. F. (2000) Cell. Microbiol. 2, 155-164
15. Glocker, E., Lange, C., Covacci, A., Bereswill, S., Kist, M., and Pahl, H. L. (1998) Infect. Immun. 66, 2346-2348
16. Bodger, K., and Crabtree, J. E. (1998) Br. Med. Bull. 54, 139-150
17. Wessler, S., Höcker, M., Fischer, W., Wang, T., Rosewicz, S., Haas, R., Wiedenmann, B., Meyer, T., and Naumann, M. (2000) J. Biol. Chem. 275, 3629-3636
18. Naumann, M., and Scheidereit, C. (1994) EMBO J. 13, 4597-4607
19. Naumann, M., Wessler, S., Bartsch, C., Wieland, B., and Meyer, T. F. (1997) J. Exp. Med. 186, 247-258
20. Lee, F., Peters, R., Dang, L., and Maniatis, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9319-9324
21. Shimada, T., Kawai, T., Takeda, K., Matsumoto, M., Inoue, J., Tatsumi, Y., Kanamaru, A., and Akira, S. (1999) Int. Immunol. 11, 1357-1362
22. Pommerantz, J., and Baltimore, D. (1999) EMBO J. 18, 6694-6704
23. Tojima, Y., Fujimoto, A., Delhase, M., Chen, Y., Hatakeyama, S., Nakayama, K., Kaneko, Y., Nimura, Y., Motoyama, N., Ikeda, K., Karin, M., and Nakanishi, M. (2000) Nature 404, 778-782
24. Hehner, S., Hofmann, T., Ushmorov, A., Dienz, O., Leung, I., Lassam, N., Scheidereit, C., Dröge, W., and Schmitz, M. (2000) Mol. Cell. Biol. 20, 2556-2568
25. Garceau, N., Kosaka, Y., Masters, S., Hambor, J., Shinkura, R., Honjo, T., and Noelle, R. (2000) J. Exp. Med. 191, 381-385
26. Lin, X., Cunningham, E., Mu, Y., Geleziunas, R., and Greene, W. (1999) Immunity 10, 271-280
27. Sells, M. A., and Chernoff, J. (1997) Trends Cell Biol. 7, 162-167
28. Zhao, Z., Manser, E., Chen, X., Chong, C., Leung, T., and Lim, L. (1998) Mol. Cell. Biol. 18, 2153-2163
29. Ninomiya-Tsuji, J., Kishimoto, K., Hiyama, A., Inoue, J., Cao, Z., and Matsumoto, K. (1999) Nature 398, 252-256
30. Takaesu, G., Kishida, S., Hiyama, A., Yamaguchi, K., Shibuya, H., Irie, K., Ninomiya-Tsuji, J., and Matsumoto, K. (2000) Mol. Cell 5, 649-658
31. Baumann, B., Weber, C., Troppmair, J., Whiteside, S., Israel, A., Rapp, U., and Wirth, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4615-4620
32. Lin, X., O'Mahony, A., Mu, Y., Geleziunas, R., and Greene, W. (2000) Mol. Cell. Biol. 20, 2933-2940
33. Sun, Z., Arendt, C., Ellmeier, W., Schaeffer, E., Sunshine, M., Gandhi, L., Annes, J., Petrzilka, D., Kupfer, A., Schwartzber, P., and Littman, D. (2000) Nature 404, 402-407
34. Coudronniere, N., Villalba, M., Englund, N., and Altman, A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3394-3399
35. O'Mahony, A., Lin, X., Geleziunas, R., and Greene, W. (2000) Mol. Cell. Biol. 20, 1170-1178
36. Zhang, S., Kovalenko, A., Cantarella, G., and Wallach, D. (2000) Immunity 12, 301-311
37. Devin, A., Cook, A., Lin, Y., Rodriguez, Y., Kelliher, M., and Liu, Z. (2000) Immunity 12, 419-429
38. Naumann, M. (2000) Biochem. Pharmacol. 60, 1109-1114
39. Orth, K., Palmer, L. E., Bao, Z. Q., Stewart, S., Rudolph, A. E., Bliska, J. B., and Dixon, J. E. (1999) Science 285, 1920-1923
40. Fackler, O., Lu, X., Frost, J., Geyer, M., Jiang, B., Luo, W., Abo, A., Alberts, A., and Peterlin, B. (2000) Mol. Cell. Biol. 20, 2619-2627
41. Bagrodia, S., and Cerione, R. A. (1999) Trends Cell Biol. 9, 350-355
42. Wu, C., Lee, S. F., Furmaniak-Kaszmierczak, E., Cote, G. P., Thomas, D. Y., and Leberer, E. (1996) J. Biol. Chem. 271, 31787-31790
43. Knaus, U. G., Morris, S., Dong, H. J., Chernoff, J., and Bokoch, G. M. (1995) Science 269, 221-223
44. Edwards, D. C., Sanders, L. C., Bokoch, G. M., and Gill, G. N. (1999) Nat. Cell Biol. 1, 253-259
45. Frost, J. A., Swantek, J. L., Stippec, S., Yin, M. J., Gaynor, R., and Cobb, M. H. (2000) J. Biol. Chem. 275, 19693-19699
46. Tuazon, P. T., Spanos, W. C., Gump, E. L., Monnig, C. A., and Traugh, J. A. (1997) Biochem. J. 36, 16059-16064
47. Naumann, M., Wessler, S., Bartsch, C., Wieland, B., Covacci, A., Haas, R., and Meyer, T. F. (1999) J. Biol. Chem. 274, 31655-31662
48. Lin, A., Minden, A., Martinetto, H., Claret, F. X., Lange-Carter, C., Mercurio, F., Johnson, G. L., and Karin, M. (1995) Science 268, 286-290
49. Tibbles, L. A., Ing, Y. L., Liefer, F., Chan, J., Iscove, N., Woodgett, J. R., and Lassam, N. J. (1996) EMBO J. 15, 7026-7035
50. Tapon, N., Nagata, K., Lamarche, N., and Hall, A. (1998) EMBO J. 17, 1395-1404

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Infect. Immun.Home page
K. Tomimori, E. Uema, H. Teruya, C. Ishikawa, T. Okudaira, M. Senba, K. Yamamoto, T. Matsuyama, F. Kinjo, J. Fujita, et al.
Helicobacter pylori Induces CCL20 Expression
Infect. Immun., November 1, 2007; 75(11): 5223 - 5232.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Rider, A. Shatrova, E. P. Feener, L. Webb, and M. Diakonova
JAK2 Tyrosine Kinase Phosphorylates PAK1 and Regulates PAK1 Activity and Functions
J. Biol. Chem., October 19, 2007; 282(42): 30985 - 30996.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Hirata, T. Ohmae, W. Shibata, S. Maeda, K. Ogura, H. Yoshida, T. Kawabe, and M. Omata
MyD88 and TNF Receptor-Associated Factor 6 Are Critical Signal Transducers in Helicobacter pylori-Infected Human Epithelial Cells
J. Immunol., March 15, 2006; 176(6): 3796 - 3803.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
Y. Hirata, S. Maeda, T. Ohmae, W. Shibata, A. Yanai, K. Ogura, H. Yoshida, T. Kawabe, and M. Omata
Helicobacter pylori Induces I{kappa}B Kinase {alpha} Nuclear Translocation and Chemokine Production in Gastric Epithelial Cells
Infect. Immun., March 1, 2006; 74(3): 1452 - 1461.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Epidemiol. Biomarkers Prev.Home page
P. Correa and B. G. Schneider
Etiology of Gastric Cancer: What Is New?
Cancer Epidemiol. Biomarkers Prev., August 1, 2005; 14(8): 1865 - 1868.
[Abstract] [Full Text] [PDF]

Home page