Janus kinase (Jak)/Stat signaling is one of the important immune and cytokine signals. Various subtypes of Jak and Stat molecules are activated by specific inflammatory stimuli.13,14 The binding of inflammatory ligand such as cytokine to its receptor induces the assembly of an active receptor associated Jak. Phosphorylated Jak leads to the activation of neighboring Jak, which provides the docking sites for Stat. Stat is phosphorylated on tyrosine and serine residues which is required for full Stat activity. Phosphorylated Stat forms dimmers and translocate to the nucleus where they bind directly to the promoter region of specific target genes involved in immune responses.15,16,17,18 In the present study, we determined whether H. pylori-induced activation of NF-κB and IL-8 production are mediated by the activation of Jak1/Stat3 in gastric epithelial AGS cells using a Jak/Stat3 specific inhibitor AG490.

A gastric epithelial cell line AGS (gastric adenocarcinoma, ATCC CRL 1739, American Type Culture Collection, Manassas, VA, USA) and H. pylori strain HP99 in Korean isolates were cultured as previously described.19 HP99 was identified as cagA+, vacA s1b, m2, iceA1 H. pylori strain.19 Tyrphostin AG490 (a Jak/Stat3 specific inhibitor) was purchased from Calbiochem (La Jolla, CA, USA). AG490 was dissolved in dimethysulfoxide (DMSO). The cells were pre-treated with AG490 (10, 20, or 40 µM final concentration) for 2 h and cultured in the presence of H. pylori for 30 min (for Jak1/Stat3 activation) and 24 h (for IL-8 levels in the medium). The control group received DMSO instead of AG490. The concentration of DMSO did not exceed 1%. In other set of experiment, AGS cells were pretreated at a final concentration of 40 µM AG490 2 h prior to H. pylori infection. The cells were cultured in the presence of H. pylori for 1 h (for NF-κB-DNA binding activity and protein levels of IκBα). IL-8 level in the medium was determined by enzyme-linked immunosorbent assay kits (Invitrogen Corporation, Carlsbad, CA, USA). For determination of protein levels of Jak1, p-Jak1, Stat, p-Stat3, IκBα, and p-IκBα, whole cell extracts (50 µg protein) were subjected to 6% SDS-PAGE and the proteins were detected with polyclonal antibodies against Jak1 (1:500, Cat. No. 3332, Cell Signaling, Beverly, MA, USA), Stat3 (1:500, Cat. No. 06-596, Upstate Biotechnology, Lake Placid, NY, USA), phospho-Jak1 (1:500, Cat. sc-16773, Santa Cruz Biotechnology, Dallas, TX, USA), phospho-Stat3 (1:500, Cat. No. 9131, Cell Signaling), pan-IκB (1:500, Cat. sc-371, Santa Cruz Biotechnology) and phospho-IκBα (1:500, Cat. No. 9241, Cell Signaling), followed by goat anti-rabbit secondary antibodies (1:2000, Cat. No. sc-2004, Santa Cruz Biotechnology) conjugated to horseradish peroxidase, which was followed by enhanced chemiluminescence (Santa Cruz Biotechnology).19 Actin served as a loading control. Electrophoretic mobility shift assay (EMSA) was performed for NF-κB-DNA binding activity using nuclear extracts as described previously.19 The statistical differences were determined using one-way ANOVA, followed by a Newman-Keul's test. All values are expressed as mean±S.E. of four different experiments. A value of p<0.05 was considered statistically significant.

Fig. 1A shows that H. pylori-induced increase in IL-8 level in the medium was inhibited by AG490 in AGS cells dose-dependently. Phospho-specific forms of Jak1 and Stat3 were increased by H. pylori which was inhibited by AG490 treatment (Fig. 1B). Total forms of Jak1 and Stat3 were not changed by H. pylori infection and AG490 treatment. These results show that AG490 efficiently inhibits Jak1/Stat3 activation in H. pylori-infected gastric epithelial AGS cells. NF-κB activation was determined by NF-κB-DNA binding activity and phophorylation of IκB by EMSA and Western blot analysis (Fig. 1C and D). AG490 suppressed phosphorylation of IκBα and NF-κB-DNA binding activity in H. pylori-infected AGS cells. Since IκBα is phosphorylated to p-IκBα by H. pylori, total IκBα was decreased in the infected cells, which was inhibited by AG490.

H. pylori-induced inflammatory signaling has been known to focuse on the activation of NF-κB with IL-8 expression.8,9,19 In the present study, we found that Jak1/Stat3 activation mediated the production of IL-8 in H. pylori-infected cells. Even though Jak/Stat signaling has been widely studied on inflammatory response in brain,20,21 relatively little studies on Jak/Stat activation in relation to gastric diseases have been reported. Lee, et al.22 reported that the phosphorylation status of H. pylori protein CagA affects the signal switch between the SHP2 (Src homology 2 domain-containing Src homology tyrosine phosphatase 2)/ERK and Jak2/Stat3 pathways through glycoprotein 130 in gastric epithelial cells. Stat3 activation mediated by non-phosphorylated CagA is dependent on Jak2 activation in AGS cells. In the present study, AG490, an inhibitor of Jak/Stat3, suppressed H. pylori-induced activation of NF-κB and IL-8 expression in AGS cells, and our recent study showed that HP99-induced activation of Jak2/Stat3 mediates the expression of monocyte chemoattractant protein-1 (MCP-1) and inducible nitric oxide synthase (iNOS) in AGS cells.23 Since we did not determine phosphorylation status of H. pylori CagA in the present study, further study should be performed to investigate the involvement of phosphorylated or non-phosphorylated CagA in Jak/Stat3-mediated NF-κB activation in gastric epithelial cells. The present results demonstrate that Jak1/Stat3 activation is an upstream signaling for NF-κB activation to induce IL-8 expression in H. pylori-infected gastric epithelial cells.

In the present study, AGS cells were infected with HP99 at bacterium/cell ratio of 300:1. Our previous study showed that Jak1/Sat3 and Jak2/Sat3 were activated at multiplicity of infection (MOI) of 300 bacteria per AGS cells,23,24 and Ritter, et al.25 demonstrated that IL-8 expression is differentially regulated by MOI of H. pylori per gastric epithelial cells; IL-8 mRNA level is elevated in response to high MOI independent of cagA and vacA gene characteristics, and only cagA+ strains significantly induce IL-8 expression by lower MOI,25 Previously, we found that both 100 and 300 MOI induced COX-2 expression26,27 whereas 300 MOI showed the activation of NF-κB and Jak1/Stat3 in HP99-infected AGS cells.24 Therefore, 100 MOI may activate Jak/Stat3 in HP99-infected cells. Further study with lower MOI is needed to determine whether Jak/Stat3 is differentially activated by MOI of H. pylori per gastric epithelial cells.

Since NF-κB activation is followed by phosphorylation and proteosomal degradation of IκBα, we determined the effect of AG490 on phosphorylation of IκBα in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 by AG490 inhibited phosphorylation of IκBα thus suppressing NF-κB activation in H. pylori-infected AGS cells. Novel finding of the present study is that the Jak1/Stat3 activation mediates the activation of NF-κB and the expression of IL-8 in H. pylori-infected AGS cells. Recently, we showed that AG490 inhibited the activation of Jak2 which is activated in HP99-infected cells.23 Therefore, Jak2 may regulate NF-κB activation in HP99-infected AGS cells. Further study should be performed to elucidate the involvement of Jak2 on H. pylori-induced NF-κB activation in gastric epithelial cells.

We earlier showed that H. pylori induced translocation of heat shock protein 90β (Hsp 90β) from cytosol to membrane in H. pylori-infected gastric epithelial cells.28 In the membrane, Hsp 90β interacts with Rac1, which activates nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) to produce reactive oxygen species (ROS). An NADPH oxidase inhibitor diphenyleneiodonium (DPI) suppressed H. pylori-induced activation of Jak1/Stat3 in AGS cells, suggesting that ROS may activate Jak1/Stat3, determined by phosphorylation of Jak1/Stat3.24 ROS are known activators for NF-κB by inducing phosphorylation of inhibitory subunit IκBα through activation of IκB kinase,29 resulting in NF-κB binding to the promoter region of IL-8 gene.30 Taken together, H. pylori induces activation of NADPH oxidase and produces ROS, resulting in the activation of both NF-κB and Jak1/Stat3 in gastric epithelial cells.

In addition, ROS activates Src kinase,31 which controls phosphorylation of H. pylori CagA32 in the infected cells. CagA activates focal adhesion kinase and Src.33 Src- dependent activation of Stat3 by tyrosine phosphorylation has been observed in renal cyst-lining cells, independently of Jak family kinases.34 Therefore, dual inhibition of Janus and Src family kinases blocks constitutively-activated Stat3 in pancreatic cancer cells.35 Even though Src kinase was not determined in the present study, Src kinase may be involved in H. pylori-induced activation of Stat3 in gastric epithelial cells. Recent study showed that thrombin-induced NF-κB activation and IL-8 release are mediated by c-Src-dependent Shc, Raf-1, and ERK pathways in lung epithelial cells.36 Therefore, Src kinase may mediate phosphorylation of IκBα and NF-κB activation in H. pylori-infected cells, which should further be studied to clarify pathologic mechanism of H. pylori-associated gastric diseases.

In the present study, we found that Jak1/Stat3 is an upstream signaling for NF-κB activation in H. pylori-infected gastric epithelial cells. Therefore, inhibition of Jak/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation by inhibiting the activation of NF-κB and suppressing inflammatory cytokine expression.