Caveolin-1 protects B6129 mice against Helicobacter pylori gastritis.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells

The function of Caveolin-1 in <i>Helicobacter pylori</i>-induced inflammation of the stomach

In dieser Studie wurde die Rolle des Caveolin-1 (Cav1) hinsichtlich der Helicobacter pylori (Hp)-induzierten Gastritis untersucht. Es wurde gezeigt, dass Cav1 vor einer verstärkten Gastritis und damit assoziierten Magenschleimhautschäden schützt sowie die durch das Hp-Toxin CagA induzierte Bildung von Stressfilamenten hemmt. Im Laufe der Infektion wurde Cav1 herunterreguliert, was als eine Strategie des Hp anzunehmen ist um so die Wirkung seiner Virulenzfaktoren in den Wirtzellen zu fördern.In this study the function of Caveolin-1 (Cav1) in Helicobacter pylori (Hp)-induced gastritis has been analyzed. It has been shown that Cav1 protects the development of enhanced gastritis and associated gastric mucosa damage and inhibits formation of stress fibers induced by Hp toxin CagA. With infection time Cav1 gene expression was down regulated. Assumably, this could be a strategy of Hp to promote the effect of its virulence factors on the host cells

Cav1 does not interact with CagA but inhibits downstream effects of CagA.

<p>(A) Cav1 does not quantitatively colocalize with <i>H. pylori</i> or CagA. AGS/Cav1, MDCK and MKN45 cells were infected with CagA-delivery proficient <i>H. pylori</i> G27 (MOI = 10) or were transiently transfected with GFP-CagA expression plasmid, followed by staining for confocal immunofluorescence microscopy; green = <i>H. pylori</i>/GFP-CagA, red = Cav1, blue = nuclei. Magnification ×630. Occasional colocalizations appear in yellow. (B) Cav1 does not directly interact with CagA. Cells were infected as in (A) for 16 h (MOI = 100), and cell lysates were incubated with rabbit polyclonal CagA and Cav1 antisera. Precipitated proteins were detected by WB. Representative CoIP experiments are shown. (C) Gentamycin protection assay. Cav1 has no impact on injection of CagA into the host cell. AGS clones were infected with <i>H. pylori</i> G27 (MOI = 500) for 2 to 24 h, extensively washed with antibiotics, and intracellular CagA was determined by WB. Representative results are shown next to the quantitative analyses. O.D. values from bands in gels were calculated as mean ± S.E. (n = 3 independent experiments); n.s. AGS/Cav1 <i>versus</i> AGS/EV.</p

Cav1 binds p120RhoGAP/DLC1 upon infection of human GC cells with <i>H. pylori</i> G27.

<p>(A) Cav1 interacts with p120RhoGAP/DLC1, a tumor suppressor and inhibitor of small GTPases associated with focal adhesions and caveolae/lipid rafts. AGS/Cav1 cells were infected with CagA-delivery competent <i>H. pylori</i> G27 (MOI = 100) for 16 h. Total cell lysates were subjected to CoIP with rabbit polyclonal Cav1-antiserum. Gels were silver stained, and peptides within a band (∼95 kDa) enriched in infected cells were analysed by MALDI-MS (see also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003251#ppat.1003251.s004" target="_blank">Table S2</a></b>). (B) Validation of MALDI-MS. Cells were infected as (A), and total cell lysates were subjected to CoIP using antibodies against Cav1 and DLC1, respectively. Representative WBs show the ∼110 kDa DLC1 protein variant 4 (DLC1v4). (C) DLC1 mRNA and protein expression. Top panel: RT-PCR gels detecting DLC1 mRNA variant 1 (full length according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003251#ppat.1003251-Low1" target="_blank">[39]</a>) in HEK293 cells, whereas DLC1v4 (short form according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003251#ppat.1003251-Low1" target="_blank">[39]</a>) was expressed in all human cancer cell lines tested. Bottom panel: Cells were transiently transfected with the expression vector pTarget-DLC1v4 (pT-DLC1v4). Representative RT-PCR and WB gels visualizing the transfected DLC1v4 mRNA and the ∼110 kDa DLC1v4 protein are shown.</p

Functional antagonism between CagA and DLC1 in gastric cancer

Helicobacter (H.) pylori-induced gastritis is a risk factor for gastric cancer (GC). Deleted-in-liver-cancer-1 (DLC1/ARHGAP7) inhibits RHOA, a downstream mediator of virulence factor cytotoxin-A (CagA) signalling and driver of consensus-molecular-subtype-2 diffuse GC. DLC1 located to enterochromaffin-like and MIST1+ stem/chief cells in the stomach. DLC1+ cells were reduced in H. pylori gastritis and GC, and in mice infected with H. pylori. DLC1 positivity inversely correlated with tumour progression in patients. GC cells retained an N-terminal truncation variant DLC1v4 in contrast to full-length DLC1v1 in non-neoplastic tissues. H. pylori and CagA downregulated DLC1v1/4 promoter activities. DLC1v1/4 inhibited cell migration and counteracted CagA-driven stress phenotypes enforcing focal adhesion. CagA and DLC1 interacted via their N- and C-terminal domains, proposing that DLC1 protects against H. pylori by neutralising CagA. H. pylori-induced DLC1 loss is an early molecular event, which makes it a potential marker or target for subtype-aware cancer prevention or therapy

Loss of Cav1 promotes recruitment of macrophages to stomachs infected with <i>H. pylori</i> SS1.

<p>(A) Differential expression of mRNAs in mouse gastric tissue upon an 11-month infection with <i>H. pylori</i> strain SS1. CT-values from RT-qPCRs on total RNA extracted from resected stomachs were normalized to beta-2-microglobulin (b2M) and are presented as mean ± S.E. (n = 15 per group); *p<0.05 as indicated by brackets and asterisks. Changes in mRNA levels were dependent on <i>H. pylori</i>-infection or genotype, respectively. (B) Cav1-KO mice infected with <i>H. pylori</i> SS1 for 11 month had pronounced infiltration of intramucosal macrophages. Immunohistochemistry (IHC) of F4/80-positive mononuclear cells. Representative images are shown.</p

Histopathology of <i>H. pylori</i> gastritis.

<p>B6129 WT and Cav1-KO mice (n = 15 per genotype) were infected with <i>H. pylori</i> SS1 for 11 months. Histopathological scores of gastritis were evaluated in H&E stainings of sections from paraffin-embedded stomach tissue. Score 0 = no, 1+ = mild, 2+ = moderate, 3+ = severe gastritis. Fisher Exact p = 0.001 (score 0 <i>versus</i> score 2+); Fisher Exact p = 0.002 (score 0 <i>versus</i> scores 1+,2+).</p

DLC1 rescues human GC cells from CagA-related adhesion defects.

<p>(A) AGS/EV cells were transiently transfected with pT-DLC1v4 plasmid for 24 h, followed by infection with CagA-delivery competent <i>H. pylori</i> G27 for additional 16 h and staining for immunofluorescence microscopy; green = actin (phalloidin), blue = nuclei. Magnification ×630. (B–C) Representative immunofluorescence images (A) are presented together with quantitative analyses of “humming bird” (B) and cell spreading (C) morphologies. Cell phenotypes were counted for at least 10 cells per field (total of 10 fields) and calculated as % ± S.E. (n = 3 experiments) of total cells; p = 0.0067 AGS/EV <i>versus</i> AGS/DLC1.</p

<i>H. pylori</i> strains down-regulate Cav1 promoter activity via activation of SREPB1.

<p>(A) <i>H. pylori</i> enhances binding of SREBP1 to one of the three sterol-responsive elements (SRE3) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003251#ppat.1003251-Bist1" target="_blank">[24]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003251#ppat.1003251-Prade1" target="_blank">[62]</a> in the proximal human Cav1 promoter. ChIP: MKN45 cells were infected with <i>H. pylori</i> G27 (MOI = 100) for 16 h, and IP was performed in total cell lysates with rabbit polyclonal acetyl-H4-histone, SREBP1 or control IgG. CT-values from qPCR of IP-ed DNA were normalized to the CT-values of input DNA and were calculated as mean ± S.E. (n = 3) of protein-DNA complex pull-down by <i>H. pylori</i> compared to empty bead and uninfected controls; *p = 0.0093 for SREBP1 and p = 0.0478 for acetyl-H4-histone, <i>H. pylori versus</i> mock. Insert: Representative WBs of nuclear extracts showing mature 68 kDa SREBP1 in the nucleus of <i>H. pylori</i> G27-infected MKN45 cells. (B) EMSA: MKN45 cells were infected with <i>H. pylori</i> G27 for 16 h, and nuclear extracts were incubated with biotin (BIO)-labelled oligonucleotide (SRE3) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003251#ppat.1003251-Prade1" target="_blank">[62]</a> from the human Cav1 promoter and an excess of unlabelled competitor oligonucleotides (SRE3-WT = wild-type; SRE3-MUT = mutant). Insert: Representative WBs of whole cell lysates showing accumulation of active 68 kDa SREBP1 in <i>H. pylori</i> G27-infected MKN45 cells. (C) <i>H. pylori</i> also regulates mRNA expression of other cognate SREBP1 target genes. CT-values from RT-qPCRs on total RNA extracted from cells infected with CagA-delivery competent G27 <i>wt</i>, CagA-deleted G27 <i>Delta cagA</i> or CagA-delivery deficient SS1 <i>H. pylori</i> strains were normalized to b2M and presented as % ± S.E. (n = 15 per group); *p = 0.0137 to 0.0196, <i>H. pylori versus</i> mock.</p

CagA evokes phosphorylation of Cav1 and Src independently of Cav1.

<p>(A–B) AGS clones were infected with CagA-delivery proficient <i>H. pylori</i> G27 (MOI = 100) for 30 min to 48 h, and cell lysates were analysed by WB. Representative WBs (A) are shown together with the quantitative summary (B). O.D. values from bands in gels were calculated as -fold ± S.E. (n = 3) compared to time point 0 (uninfected cells). n.s. AGS/Cav1 <i>versus</i> AGS/EV.</p