<i>Helicobacter pylori</i> Initiates a Mesenchymal Transition through ZEB1 in Gastric Epithelial Cells

<div><p>Chronic <i>Helicobacter pylori</i> infection provokes an inflammation of the gastric mucosa, at high risk for ulcer and cancer development. The most virulent strains harbor the <i>cag</i> pathogenicity island (<i>cag</i>PAI) encoding a type 4 secretion system, which allows delivery of bacterial effectors into gastric epithelial cells, inducing pro-inflammatory responses and phenotypic alterations reminiscent of an epithelial-to-mesenchymal transition (EMT). This study characterizes EMT features in <i>H. pylori</i>-infected gastric epithelial cells, and investigates their relationship with NF-κB activation. Cultured human gastric epithelial cell lines were challenged with a <i>cag</i>PAI<i>+ H. pylori</i> strain or <i>cag</i> isogenic mutants. Morphological changes, epithelial and mesenchymal gene expression and EMT-related microRNAs were studied. <i>H. pylori</i> up-regulates mesenchymal markers, including ZEB1. This transcription factor is prominently involved in the mesenchymal transition of infected cells and its up-regulation depends on <i>cag</i>PAI and NF-κB activation. ZEB1 expression and NF-κB activation were confirmed by immunohistochemistry in gastric mucosa from <i>cag</i>PAI<i>+ H. pylori</i>-infected patients. Gastric epithelial cell lines express high miR-200 levels, which are linked to ZEB1 in a reciprocal negative feedback loop and maintain their epithelial phenotype in non-infected conditions. However, miR-200b/c were increased upon infection, despite ZEB1 up-regulation and mesenchymal morphology. In the miR-200b-200a-429 cluster promoter, we identified a functional NF-κB binding site, recruiting NF-κB upon infection and trans-activating the microRNA cluster transcription. In conclusion, in gastric epithelial cells, <i>cag</i>PAI+ <i>H. pylori</i> activates NF-κB, which transactivates ZEB1, subsequently promoting mesenchymal transition. The unexpected N-FκB-dependent increase of miR-200 levels likely thwarts the irreversible loss of epithelial identity in that critical situation.</p> </div

Kinetics of changes in IL-8 induction (A), ZEB1 and pri-miR-200b (B) and E-cadherin and vimentin (C) expressions upon infection.

<p>Cells were infected with either wt <i>H. pylori</i> (WT) or its <i>cagA</i>-deletion isogenic mutant (ΔCagA) at MOI 100 bacteria/cells for the indicated period of times. Bars indicate the fold changes of the individual genes upon infection (mean of duplicates ± SD of RNA expression normalized to HPRT1 and compared to NI).</p

Changes in mesenchymal and epithelial gene expression and miR-200 levels. 10 days post-infection with cagPAI+ H. pylori (Hp WT) or the isogenic CagA-deficient strain.

<p>48 hrs post-infection at a MOI 100, infected and non-infected cells were trypsinized and subcultured for 10 days in a 6-well plate starting at an initial cell density of 2,000 cells/well. The culture medium was changed every other day. Data represent mean ± SD of RTqPCR results of the individual genes or miRNAs relative to HPRT1 or snoR25, respectively, and compared to non infected cells; n = 4; *: p-value <0.05, **: p-value <0.01, ***: p-value <0.001.</p

MiR-200 regulate ZEB1 expression in basal conditions and are up-regulated by <i>H. pylori</i>.

<p>(A) Cell morphology observed by phase contrast microscopy of AGS cells treated for 5 day with 100 nM antisense (as200b/c) or scrambled (sc200) oligonucleotides (scale bar, 20 µm). (B) ZEB1 immunofluorescence in the same conditions (scale bar, 20 µm) (C) RT-qPCR data of ZEB1 mRNA in the same conditions. Bars represent the mean ± SD of ZEB1 mRNA relative to HPRT mRNA compared to non transfected cells (n = 5; * p<0.05). (D) ZEB1 and tubulin immunoblots in the same conditions. (E) RT-qPCR data of mature miR-200b and -200c in AGS cells infected or not (NI) with either wt <i>H. pylori</i> (WT) or isogenic mutants (ΔCagA, ΔCagE) at MOI 100 bacteria/cells for 24 h. Bars indicate mean ± SD of miRNA expression normalized to U6 snRNA and compared to NI (n = 6; ** p<0.01). (F) RT-qPCR data of primary miR-200b-200a-429 transcript in the same infection conditions. Bars indicate mean ± SD of pri-miRNA expression normalized to HPRT1 and compared to NI (n = 4; * p<0.05, *** p<0.01).</p

<i>H. pylori</i> up-regulates ZEB1 in gastric epithelial cells.

<p>(A) RT-qPCR data of ZEB1 mRNA upon 24 h infection with wild type <i>H. pylori</i> (Hp WT) or its isogenic mutants deleted either for <i>cagA</i> (Hp Δ<i>cag</i>A) or <i>cagE</i> (Hp Δ<i>cag</i>E); bars represent the mean ± SD of ZEB1 mRNA relative to HPRT mRNA compared to non infected cells (NI) (n = 5; * p<0.05). (B) ZEB1 immunofluorescence in non-infected cells (NI) or upon infection as in (A); bar, 40 µm. (C) Cell morphology observed by phase contrast microscopy of AGS cells transfected with siZEB1 or control siRNA (20 nM) prior infection as in (A). Bar, 40 µm. (D) ZEB1 or α-tubulin immunoblots in cells treated with siZEB1 or control siRNA and infected as in (A).</p

<i>H. pylori</i>, ZEB1, p65 NF-κB, E-cadherin immunostaining and miR-200b <i>in situ</i> hybridization in non infected human gastric mucosa (left pannels) or mucosa infected with <i>cag</i>PAI+ <i>H. pylori</i> (right pannels).

<p>Images are representatives of the detection by immunohistochemistry coupled to peroxydase activity (in brown) of <i>H. pylori</i> in the lumen of gastric glands at the apical surface of gastric epithelial cells, which display an intense ZEB1 and p65 expression mainly in the nucleus, despite a similar E-cadherin expression and localization at cell/cell junction in the <i>H. pylori</i> infected specimen and the non-infected one. MiR-200b detected by ISH coupled to phosphatase alkaline activity (in dark blue) is highly expressed in gastric glands of <i>H. pylori</i>–infected case. Typical images of the same case out of three infected patients and the same case out of three uninfected cases are shown. Bar, 50 µm.</p

Changes in expression of mesenchymal and epithelial markers in AGS, MKN74 and NCI-N87 cells infected for 24 h with the H. pylori strain 26695.

<p>Data represent RT-qPCR results of the individual genes normalized to that of HPRT and compared to non infected cells (mean, n = 3, *p<0.05, **p<0.01, ***p<0.001).</p

NF-κB-dependent mesenchymal phenotype of infected AGS cells.

<p>In all experiments, AGS cells were infected or not (NI) with <i>cagPAI+ H. pylori</i> (WT) at MOI 100 bacteria/cells for 24 h. (A) Activities of SV40 promoter (pGL3-p), or <i>miR-200b-200a-429</i> promoter wild type (pGL3-prom200b) or mutated on the NF-κB site (pGL3-prom200b mut); bars represent mean ± SD of relative luciferase activities of each promoter reporter normalized to that of NI pGL3-p transfected cells (n = 3; ** p<0.01). (B) NF-κB activation upon infection (WT) in cells transfected either with pEGFP or with pEGFP-IκB. Bars represent mean ± SD of relative NF-κB reporter luciferase activity compared to NI pEGFP-transfected cells (n = 3; *p<0.05). (C) Cell morphology observed by phase contrast microscopy, in the same conditions. Bar, 20 µm. (D) RT-qPCR data of ZEB1 and pri-miR-200b-200a-429 in pEGFP- or pEGFP-IκB-transfected cells. Bars indicate mean ± SD of RNA expression normalized to HPRT1 and compared to NI (n = 3, * p<0.05, *** p<0.001). (E) <i>MiR-200b-200a-429</i> promoter activities measured as in (A) in cells transfected either with pEGFP or with pEGFP-IκB. (F) Chromatin immunoprecipitation assays using anti-NF-κB antibody on the promoters of miR-200b (prom200b), IL-8 (promIL-8) or ZEB1 (promZEB1). Bars represent NF-κB enrichment on a given promoter in either uninfected or infected cells, calculated as the following ratio: 2^{−ΔCt IPNF-κB}/2^{−ΔCt controlIP}, with ΔCt = Ct IP (with NF-κB antibody or without (control)) – Ct input (input corresponds to chromatin before immunoprecipitation).</p