TIFA has dual functions in Helicobacter pylori-induced classical and alternative NF-κB pathways.

Helicobacter pylori infection constitutes one of the major risk factors for the development of gastric diseases including gastric cancer. The activation of nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) via classical and alternative pathways is a hallmark of H. pylori infection leading to inflammation in gastric epithelial cells. Tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA) was previously suggested to trigger classical NF-κB activation, but its role in alternative NF-κB activation remains unexplored. Here, we identify TRAF6 and TRAF2 as binding partners of TIFA, contributing to the formation of TIFAsomes upon H. pylori infection. Importantly, the TIFA/TRAF6 interaction enables binding of TGFß-activated kinase 1 (TAK1), leading to the activation of classical NF-κB signaling, while the TIFA/TRAF2 interaction causes the transient displacement of cellular inhibitor of apoptosis 1 (cIAP1) from TRAF2, and proteasomal degradation of cIAP1, to facilitate the activation of the alternative NF-κB pathway. Our findings therefore establish a dual function of TIFA in the activation of classical and alternative NF-κB signaling in H. pylori-infected gastric epithelial cells.

A20 undermines alternative NF-κB activity and expression of anti-apoptotic genes in Helicobacter pylori infection.

A hallmark of infection by the pathogen Helicobacter pylori, which colonizes the human gastric epithelium, is the simultaneous activation of the classical and alternative nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, underlying inflammation and cell survival. Here, we report that the classical NF-κB target gene product A20 contributes to the negative regulation of alternative NF-κB signaling in gastric epithelial cells infected by H. pylori. Mechanistically, the de novo synthesized A20 protein interacts with tumor necrosis factor receptor-associated factor-interacting protein with forkhead-associated domain (TIFA) and thereby interferes with the association of TIFA with the NIK regulatory complex. We also show that alternative NF-κB activity contributes to the up-regulation of anti-apoptotic genes, such as baculoviral IAP repeat containing 2 (BIRC2), BIRC3 and B-cell lymphoma 2-related protein A1 (BCL2A1) in gastric epithelial cells. Furthermore, the observed over-expression of RelB in human gastric biopsies with type B gastritis and RelB-dependent suppression of apoptotic cell death emphasize an important role of the alternative NF-κB pathway in H. pylori infection.

CEACAMs interaction with Helicobacter pylori HopQ supports the type 4 secretion system-dependent activation of non-canonical NF-κB.

Helicobacter pylori infection represents a major risk factor for the development of gastric diseases and gastric cancer. The capability of H. pylori to inject the virulence factor cytotoxin-associated gene A (CagA) depends on a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI). Further, infection by H. pylori activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in a T4SS-dependent manner but CagA-independent manner. Here we investigated the role of host cell receptors carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) and the bacterial adhesin HopQ in the activation of non-canonical NF-κB and CagA translocation into gastric epithelial cells. AGS cells express six of twelve CEACAMs found in humans. In HeLa cells, only CEACAM19 is expressed. We showed that deletion of hopQ attenuates the activation of non-canonical NF-κB only in AGS but not in HeLa cells. CagA translocation was in both cell lines affected by HopQ depletion, although to a much lesser extent in HeLa cells. Moreover, we observed a possible redundancy between the three HopQ-binding CEACAMs 1, 5 and 6 and their capacity to support non-canonical NF-κB activation. Our results illustrate that the interaction between HopQ and CEACAMs could promote the efficiency of the T4SS.

HopQ impacts the integrin α5ß1-independent NF-κB activation by Helicobacter pylori in CEACAM expressing cells.

Helicobacter pylori infection persists in more than half of the world's population and represents a risk factor for peptic ulcer disease and gastric cancer. Virulent strains of H. pylori carry a cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) with the capability to inject the effector protein cytotoxin-associated gene A (CagA) into eukaryotic cells. Colonisation of the gastric epithelium by H. pylori provokes direct activation of the proinflammatory and survival factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We investigated the impact of host cell receptor integrin α5ß1 and the bacterial adhesin HopQ on the NF-κB activation. We found that H. pylori induced early T4SS-dependent, but CagA-independent canonical NF-κB signalling in polarized, apical infected NCI-N87 cells. Integrin-dependent CagA translocation was hardly detectable, as integrin ß1 was sparsely located at the apical surface of polarized NCI-N87 cells. Knockdown experiments indicated that integrin α5ß1 and integrin linked kinase (ILK) were dispensable for NF-κB activation in H. pylori infection. Thus, there exists no common mechanism, which mediates integrin α5ß1-dependent H. pylori-triggered CagA translocation and the activation of NF-κB. Further, we report that H. pylori adhesin HopQ, which binds to a specific subset of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), promotes canonical NF-κB activation in AGS and NCI-N87 cells, but not in HeLa cells, which are devoid of these CEACAMs. Noteworthy, these effects were not mediated by reduced adhesion, indicating additional functions of HopQ.

Pathogenesis of Helicobacter pylori infection.

Helicobacter pylori is estimated to infect more than half of the worlds human population and represents a major risk factor for chronic gastritis, peptic ulcer disease, MALT lymphoma, and gastric adenocarcinoma. H. pylori infection and clinical consequences are controlled by highly complex interactions between the host, colonizing bacteria, and environmental parameters. Important bacterial determinants linked with gastric disease development include the cag pathogenicity island encoding a type IV secretion system (T4SS), the translocated effector protein CagA, vacuolating cytotoxin VacA, adhesin BabA, urease, serine protease HtrA, secreted outer membrane vesicles, and many others. The high quantity of these factors and allelic changes in the corresponding genes reveals a sophisticated picture and problems in evaluating the impact of each distinct component. Extensive work has been performed to pinpoint molecular processes related to H. pylori-triggered pathogenesis using Mongolian gerbils, mice, primary tissues, as well as novel in vitro model systems such as gastroids. The manipulation of host signaling cascades by the bacterium appears to be crucial for inducing pathogenic downstream activities and gastric disease progression. Here, we review the most recent advances in this important research area.

MEKK3 and TAK1 synergize to activate IKK complex in Helicobacter pylori infection.

Helicobacter pylori colonises the gastric epithelial cells of half of the world's population and represents a risk factor for gastric adenocarcinoma. In gastric epithelial cells H. pylori induces the immediate early response transcription factor nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) and the innate immune response. We show that H. pylori induces in a type IV secretion system-dependent (T4SS) and cytotoxin associated gene A protein (CagA)-independent manner a transient activation of the inhibitor of NF-κB (IκBα) kinase (IKK)-complex. IKKα and IKKß expression stabilises the regulatory IKK complex subunit NF-κB essential modulator (NEMO). We provide evidence for an intimate mutual control of the IKK complex by mitogen-activated protein kinase kinase kinase 3 (MEKK3) and transforming growth factor ß activated kinase 1 (TAK1). TAK1 interacts transiently with the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6). Protein modifications in the TAK1 molecule, e.g. TAK1 autophosphorylation and K63-linked ubiquitinylation, administer NF-κB signalling including transient recruitment of the IKK-complex. Overall, our data uncover H. pylori-induced interactions and protein modifications of the IKK complex, and its upstream regulatory factors involved in NF-κB activation.

Harnessing gastrointestinal organoids for cancer therapy.

Gastrointestinal organoids have emerged as a model system that authentically recapitulates the in vivo situation. Despite biomedical and technical challenges, self-assembled 3D structures derived from pluripotent stem cells or healthy and diseased tissues have proved to be invaluable tools for cancer drug discovery, disease modeling, and studying infection with carcinogenic pathogens.

Ca2+/calmodulin-dependent kinase II contributes to inhibitor of nuclear factor-kappa B kinase complex activation in Helicobacter pylori infection.

Helicobacter pylori, a class I carcinogen, induces a proinflammatory response by activating the transcription factor nuclear factor-kappa B (NF-κB) in gastric epithelial cells. This inflammatory condition could lead to chronic gastritis, which is epidemiologically and biologically linked to the development of gastric cancer. So far, there exists no clear knowledge on how H. pylori induces the NF-κB-mediated inflammatory response. In our study, we investigated the role of Ca(2+) /calmodulin-dependent kinase II (CAMKII), calmodulin, protein kinases C (PKCs) and the CARMA3-Bcl10-MALT1 (CBM) complex in conjunction with H. pylori-induced activation of NF-κB via the inhibitor of nuclear factor-kappa B kinase (IKK) complex. We use specific inhibitors and/or RNA interference to assess the contribution of these components. Our results show that CAMKII and calmodulin contribute to IKK complex activation and thus to the induction of NF-κB in response to H. pylori infection, but not in response to TNF-α. Thus, our findings are specific for H. pylori infected cells. Neither the PKCs α, δ, θ, nor the CBM complex itself is involved in the activation of NF-κB by H. pylori. The contribution of CAMKII and calmodulin, but not PKCs/CBM to the induction of an inflammatory response by H. pylori infection augment the understanding of the molecular mechanism involved and provide potential new disease markers for the diagnosis of gastric inflammatory diseases including gastric cancer.

Helicobacter pylori regulates TIFA turnover in gastric epithelial cells.

The human pathogen Helicobacter pylori induces a strong inflammatory response in gastric mucosa manifested by the recruitment of neutrophils and macrophages to the places of infection, and by changes in epithelial integrity and function. At the molecular level, this innate immune response is essentially dependent on the activation of NF-κB transcription factors regulating the expression of chemotactic factors, e.g., IL-8. Recently, it has been demonstrated that the NF-κB signaling pathway is triggered by the bacterial heptose metabolites, which activate the host ALPK1-TIFA axis. TIFA has been suggested to promote oligomerization and activity of the E3 ubiquitin ligase TRAF6, which further stimulates TAK1-IKK signaling. Here, we demonstrate that ALPK1-dependent TIFA activation in H. pylori-infected gastric epithelial cells is followed in time by a decline in TIFA levels, and that this process is impeded by inhibitors of the proteasomal and lysosomal degradation. According to our data, TRAF2, TRAF6, TAK1 or NEMO are not required for TIFA degradation. Additionally, H. pylori promotes the interaction of TIFA with free polyubiquitin as well as with optineurin, TAX1BP1 and LAMP1, which are known protein adaptors involved in intracellular trafficking to lysosomes.

Helicobacter pylori-induced NF-κB: trailblazer for gastric pathophysiology.

NF-κB signaling pathways, induced by a variety of triggers, play a key role in regulating the expression of genes involved in the immune response and cellular responses to stress. The human pathogen Helicobacter pylori induces classical and alternative NF-κB signaling pathways via its effector ADP-L-glycero-ß-D-manno-heptose (ADP-heptose). We review H. pylori- and NF-κB-dependent alterations in cellular processes and associated maladaptation leading to deleterious gastric pathophysiology that have implications for the diagnosis and treatment of gastric diseases. Therapeutic options for gastric cancer (GC) include clinically relevant small molecule inhibitors of NF-κB and epigenetic therapy approaches. In this context, gastric organoid biobanks originated from patient material, represent a valuable platform for translational applications to predict patient responses to chemotherapy, with a view to personalized medicine.

Human gastric fibroblasts ameliorate A20-dependent cell survival in co-cultured gastric epithelial cells infected by Helicobacter pylori.

Crosstalk within the gastric epithelium, which is closely in contact with stromal fibroblasts in the gastric mucosa, has a pivotal impact in proliferation, differentiation and transformation of the gastric epithelium. The human pathogen Helicobacter pylori colonises the gastric epithelium and represents a risk factor for gastric pathophysiology. Infection of H. pylori induces the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is involved in the pro-inflammatory response but also in cell survival. In co-cultures with human gastric fibroblasts (HGF), we found that apoptotic cell death is reduced in the polarised human gastric cancer cell line NCI-N87 or in gastric mucosoids during H. pylori infection. Interestingly, suppression of apoptotic cell death in NCI-N87 cells involved an enhanced A20 expression regulated by NF-κB activity in response to H. pylori infection. Moreover, A20 acts as an important negative regulator of caspase-8 activity, which was suppressed in NCI-N87 cells during co-culture with gastric fibroblasts. Our results provide evidence for NF-κB-dependent regulation of apoptotic cell death in cellular crosstalk and highlight the protective role of gastric fibroblasts in gastric epithelial cell death during H. pylori infection.

NF-kappaB-inducing kinase in cancer.

Dysregulation of the alternative NF-κB signaling has severe developmental consequences that can ultimately lead to oncogenesis. Pivotal for the activation of the alternative NF-κB pathway is the stabilization of the NF-κB-inducing kinase (NIK). The aim of this review is to focus on the emerging role of NIK in cancer. The documented subversion of NIK in cancers highlights NIK as a possible therapeutic target. Recent studies show that the alterations of NIK or the components of its regulatory complex are manifold including regulation on the transcript level, copy number changes, mutations as well as protein modifications. High NIK activity is associated with different human malignancies and has adverse effects on tumor patient survival. We discuss here research focusing on deciphering the contribution of NIK towards cancer development and progression. We also report that it is possible to engineer inhibitors with high specificity for NIK and describe developments in this area.

Expression and upregulation of cathepsin S and other early molecules required for antigen presentation in activated hepatic stellate cells upon IFN-gamma treatment.

Hepatic stellate cells (HSCs) have been shown to be able to activate T-cells and upregulate expression of surface molecules essential for this process, when treated with IFN-gamma. But little is known about the early molecules expressed by activated hepatic stellate cells under the same treatment. In this study, we investigate the effect of IFN-gamma on the transcription and expression of these early molecules in hepatic stellate cells. We show on the molecular level that activated rat hepatic stellate cells express the class II transactivator, the invariant chain (CD74), the MHC class II molecules, as well as cathepsin S, all of which are known to be responsible for the initial steps of successful antigen presentation. The mRNA and the protein expression level of these molecules is upregulated by IFN-gamma. Importantly, IFN-gamma increases cathepsin S activity, suggesting a possible involvement of this protease in CD74 processing. Our data also show that not only can the HSCs take up antigenic proteins, they can also process them. Our comparative study indicates that the rat HSC-T6 cell line displays sufficient similarity to the activated rat HSCs in order to serve as a model for in vitro studies on the molecular mechanisms of inflammatory response.

Glial fibrillary acidic protein splice variants in hepatic stellate cells--expression and regulation.

The glial fibrillary acidic protein (GFAP) is traditionally used as a marker for astrocytes of the brain, and more recently for the hepatic stellate cells (HSCs) of the liver. Several GFAP splice variants have been previously reported in the astrocytes of the CNS and in the non-myelinating Schwann cells of the PNS. In this study, we investigate whether GFAP splice variants are present in the HSCs and their expression as a function of HSCs activation. Furthermore, the regulation of these transcripts upon treatment with interferon gamma (IFN-gamma) will be explored. Using semi-quantitative RT-PCR and real-time PCR, we examine the expression and regulation of GFAP splice variants in HSCs as well as their respective half-life. We discover that most of the GFAP splice variants (GFAPalpha, beta, delta, epsilon, and kappa) found in the neural system are also expressed in quiescent and culture-activated primary HSCs. Interestingly, GFAPalpha is the predominant form in quiescent and culture-activated primary HSCs, while GFAPbeta predominates in the SV40-immortalized activated HSC-T6. GFAP delta, epsilon, and kappa have similar half-lives of 10 hours, while GFAPbeta has a half-life of 17 hours. Treatment of HSC-T6 with IFN-gamma results in a significant 1.29-fold up-regulation of GFAPalpha; whereas the level of the other transcripts remains unchanged. In summary, GFAPalpha, beta, delta, epsilon, and kappa are present in HSCs. They are differentially regulated on the transcription level, implying a role of the 5' and 3' untranslated regions.

NEMO Links Nuclear Factor-κB to Human Diseases.

The nuclear factor (NF)-κB essential modulator (NEMO) is a key regulator in NF-κB-mediated signaling. By transmitting extracellular or intracellular signals, NEMO can control NF-κB-regulated genes. NEMO dysfunction is associated with inherited diseases such as incontinentia pigmenti (IP), ectodermal dysplasia, anhidrotic, with immunodeficiency (EDA-ID), and some cancers. We focus on molecular studies, human case reports, and mouse models emphasizing the significance of NEMO molecular interactions and modifications in health and diseases. This knowledge opens new opportunities to engineer suitable drugs that may putatively target precise NEMO functions attributable to various diseases, while leaving other functions intact, and eliminating cytotoxicity. Indeed, with the advent of novel gene editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9, treating some inherited diseases may in the long run, become a reality.

Pathogen-induced ubiquitin-editing enzyme A20 bifunctionally shuts off NF-κB and caspase-8-dependent apoptotic cell death.

The human pathogen Helicobacter pylori infects more than half of the world's population and is a paradigm for persistent yet asymptomatic infection but increases the risk for chronic gastritis and gastric adenocarcinoma. For successful colonization, H. pylori needs to subvert the host cell death response, which serves to confine pathogen infection by killing infected cells and preventing malignant transformation. Infection of gastric epithelial cells by H. pylori provokes direct and fast activation of the proinflammatory and survival factor NF-κB, which regulates target genes, such as CXCL8, BIRC3 and TNFAIP3. However, it is not known how H. pylori exploits NF-κB activation and suppresses the inflammatory response and host apoptotic cell death, in order to avert the innate immune response and avoid cell loss, and thereby enhance colonization to establish long-term infection. Here we assign for the first time that H. pylori and also Campylobacter jejuni-induced ubiquitin-editing enzyme A20 bifunctionally terminates NF-κB activity and negatively regulates apoptotic cell death. Mechanistically, we show that the deubiquitinylase activity of A20 counteracts cullin3-mediated K63-linked ubiquitinylation of procaspase-8, therefore restricting the activity of caspase-8. Interestingly, another inducible NF-κB target gene, the scaffold protein p62, ameliorates the interaction of A20 with procaspase-8. In conclusion, pathogen-induced de novo synthesis of A20 regulates the shut-off of the survival factor NF-κB but, on the other hand, also impedes caspase-8-dependent apoptotic cell death so as to promote the persistence of pathogens.

GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line.

AIM: The GFAP was traditionally considered to be a biomarker for neural glia (mainly astrocytes and non-myelinating Schwann cells). Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells (HSC). In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS: The GFAP-lacZ transgene was transfected into various cell lines (HSC, hepatocyte, and other non-HSC cell types). The transgene expression specificity was determined by X-gal staining of the beta-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-beta1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS: The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-beta1 by upregulation in a dose- and time-dependent manner, similar to the endogenous GFAP. CONCLUSION: In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.