Gunter Maubach

TGF-β1 down-regulates connexin 43 expression and gap junction intercellular communication in rat hepatic stellate cells

Intercellular communication is an important tool used by the cells to effectively regulate concerted responses. Hepatic stellate cells (HSCs) communicate to each other through functional gap junctions composed of connexin 43 (Cx43) proteins. We show that exogenous human TGF-beta1 (hTGF-beta1), a pro-fibrotic stimulus, decreases Cx43 mRNA and protein in a rat HSC cell line and primary HSCs. Furthermore, hTGF-beta1 increases the phosphorylation of Cx43 at serine 368. These effects lead to a decrease in the gap junction intercellular communication between the HSCs, as shown by gap-FRAP analysis. We also observe the binding of Snai1, from the nuclear extract of HSCs, to a Snai1 consensus sequence in the Cx43 promoter. In the same context, Snai1 siRNA transfection results in an up-regulation of Cx43 suggesting that TGF-beta1 may regulate Cx43 via Snai1. In addition, we demonstrate that the knockdown of Cx43 by siRNA transfection results in a slower proliferation of HSCs. These findings illuminate a new effect of TGF-beta1 in HSCs, namely the regulation of intercellular communication by affecting the expression level and the phosphorylation state of Cx43 through Snai1 signaling.

Nuclear Cathepsin F Regulates Activation Markers in Rat Hepatic Stellate Cells

Activation of hepatic stellate cells during liver fibrosis is a major event facilitating an increase in extracellular matrix deposition. The up-regulation of smooth muscle alpha-actin and collagen type I is indicative of the activation process. The involvement of cysteine cathepsins, a class of lysosomal cysteine proteases, has not been studied in conjunction with the activation process of hepatic stellate cells. Here we report a nuclear cysteine protease activity partially attributed to cathepsin F, which co-localizes with nuclear speckles. This activity can be regulated by treatment with retinol/palmitic acid, known to reduce the hepatic stellate cell activation. The treatment for 48 h leads to a decrease in activity, which is coupled to an increase in cystatin B and C transcripts. Cystatin B knockdown experiments during the same treatment confirm the regulation of the nuclear activity by cystatin B. We demonstrate further that the inhibition of the nuclear activity by E-64d, a cysteine protease inhibitor, results in a differential regulation of smooth muscle alpha-actin and collagen type I transcripts. On the other hand, cathepsin F small interfering RNA transfection leads to a decrease in nuclear activity and a transcriptional down-regulation of both activation markers. These findings indicate a possible link between nuclear cathepsin F activity and the transcriptional regulation of hepatic stellate cell activation markers.

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

Helicobacter pylori colonises the gastric epithelial cells of half of the worlds 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 IKKβ 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 β 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.

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.

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 Ca2+/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.

Pathogenesis ofHelicobacter pyloriinfection

The clinical outcome of Helicobacter pylori infection is determined by a complex scenario of interactions between the bacterium and the host. The main bacterial factors associated with colonization and pathogenicity comprise outer membrane proteins including BabA, SabA, OipA, AlpA/B, as well as the virulence factors CagA in the cag pathogenicity island (cagPAI) and the vacuolating cytotoxin VacA. The multitude of these proteins and allelic variation makes it extremely difficult to test the contribution of each individual factor. Much effort has been put into identifying the mechanism associated with H. pylori‐associated carcinogenesis. Interaction between bacterial factors such as CagA and host signal transduction pathways seems to be critical for mediating the induction of membrane dynamics, actin‐cytoskeletal rearrangements and the disruption of cell‐to‐cell junctions as well as proliferative, pro‐inflammatory and antiapoptotic nuclear responses. An animal model using the Mongolian gerbil is a useful system to study the gastric pathology of H. pylori infection.

HopQ impacts the integrin α5β1-independent NF-κB activation by Helicobacter pylori in CEACAM expressing cells

Helicobacter pylori infection persists in more than half of the worlds 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 α5β1 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 β1 was sparsely located at the apical surface of polarized NCI-N87 cells. Knockdown experiments indicated that integrin α5β1 and integrin linked kinase (ILK) were dispensable for NF-κB activation in H. pylori infection. Thus, there exists no common mechanism, which mediates integrin α5β1-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.

Constitutive activation of nuclear factor kappa B-inducing kinase counteracts apoptosis in cells with rearranged mixed lineage leukemia gene

Chromosomal rearrangements and translocations of the mixed-lineage leukemia 1 (MLL1, 11q23) gene are responsible for 5–10% of childhood and adult acute myeloid leukemia cases [1]. A frequent alteration of the MLL gene are reciprocal translocations giving rise to MLL fusion oncoprotein (Supplementary Figure S1a) that exhibit a loss of MLLs H3K4 methyltransferase activity affecting histone modifications and thereby gene transcription [1]. One prominent feature of MLL-rearranged leukemias is the upregulation of the later HOX cluster genes and MEIS1 genes, which expression is crucial in developmental processes like hematopoiesis [1]. In general, the prognosis for these patients is very poor compared to other leukemias [2]. A recent study demonstrated that the canonical nuclear factorκB (NF-κB) signaling contributes to MLL fusion proteindependent ectopic HOX expression, promotion of proliferation, survival, and differentiation arrest of leukemic cells [3]. Remarkably, some hematological cancers such as multiple myeloma, T-cell leukemia, or Hodgkin ReedSternberg cells show an increased activity of the canonical NF-κB, which might, in some cases, be accompanied by a constitutively active non-canonical NF-κB pathway [4–6]. Due to the discrete activation of the non-canonical NF-κB pathway by a subset of TNF family receptors, it constitutes an attractive therapeutic target [7]. Therefore, we investigated for the first time the contribution of the non-canonical NF-κB signaling to the survival of MLL-rearranged leukemia cells after chemotherapeutic drug treatment. To overcome the inherent genetic heterogeneity of MLL patient material, we used primary human CD34+ cord blood cell line transduced with MLL-AF9 fusion cDNA [8]. As reported by Wei et al. [8], this modified cell line displays a strong similarity to the gene expression profile of cells withMLL-AF9 fusion obtained from patients with AML. To fully assess the non-canonical NF-κB activity in AML with MLL gene rearrangement, we compared transduced cells to closely related, yet non-transduced counterparts, the generally accepted human CD34+ blood stem/progenitor cells [9]. In addition, CD34+ MLL-AF9 cells were compared to another primary human CD34+ cord blood cell line carrying an AML1-ETO rearrangement [10] to evaluate a different type of gene translocation driving AML, but with a better prognosis than MLL-AF9. Firstly, we analyzed lymphotoxin-β receptor (LTβR) expression, recently reported to be responsible for the aberrant activity of the non-canonical NF-κB in some molecular subtypes of T-cell acute lymphoblastic leukemia [11], and NF-κB-inducing kinase (NIK) a main regulator of the non-canonical NF-κB pathway. We demonstrated that LTβR indeed exhibits significantly elevated expression only in cells carrying MLL-AF9 rearrangement (Fig. 1a), but is absent in control CD34+ cells and those with AML1-ETO rearrangement. Induced self-association due to high abundance of LTβR can lead to constitutive NF-κB signaling [11], therefore we studied further the expression of noncanonical NF-κB components. The analysis revealed that cells with MLL-AF9 rearrangement exhibit a constitutively stabilized NIK possessing the activating phosphorylation (T559). Further, phosphorylated p100 subsequently processed to p52 was observed (Fig. 1a). Noteworthy in this context, CD34+MLL-AF9 cells displayed in contrast to the control cells high expression of RelB (Fig. 1a), which is required for the transcriptionally active non-canonical NF* Michael Naumann naumann@med.ovgu.de

NF-κB-regulated ubiquitin-editing enzyme A20 paves the way for infection persistency

Infection by microbial pathogens modulates apoptotic cell death of the host cells as one of the host immune defense mechanisms. Counteracting the host apoptotic cell death could be directed by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), which is induced upon infection and triggers the host inflammatory response against pathogens. The NF-kB family of five transcription factors directs nuclear responses leading to many cellular processes including inflammation, proliferation or survival. Apoptotic cell death of infected cells benefits the host as it limits the infection through the elimination of infected cells, in the form of apoptotic bodies, by professional phagocytes and thereby makes microbial antigens available for presentation by major histocompatibility complex class I or II molecules to induce a protective immune response. Bacterial pathogens however tend to suppress host cell death during the initial stages of infection for colonization and later, induce host cell death which is necessary not only as a source of nutrients from the cell debris, but also for the further propagation of the bacteria. For these reasons in fact, bacteria have evolved sophisticated strategies to manipulate the host cell death pathways. Notably, these evasion strategies originate from the synthesis and translocation of bacterial effector proteins to interfere with the NF-kB activation. An early report from Yanai and colleagues showed the involvement of NF-kB in promoting the survival of cells infected with the human gastric pathogen Helicobacter pylori, opening up the possibility that rather than utilizing self-synthesized effector molecules, bacteria can benefit from infection-activated NF-kB. Our latest report showed specifically the upregulation of NFkB-dependent A20 after infections by H. pylori and Campylobacter jejuni. A20 (also known as Tumor Necrosis Factor Alpha-Induced Protein 3, TNFAIP3) is a unique zinc finger protein possessing a deubiquitinylase activity as well as an E3 ubiquitin ligase activity. Both activities are required to terminate NF-kB activation in a negative feedback loop, explaining the transient nature of an inflammatory response. In H. pylori-infected gastric epithelial cells, A20 inhibits not only prosurvival NF-kB signaling, preventing the expression of proinflammatory IL-8, but also apoptotic cell death. Specifically for the latter, we provided evidence demonstrating that A20 interferes with the activation of procaspase-8 and interestingly, another NF-kB target p62 (sequestosome-1) is important for this interaction of A20 with procaspase-8 (Fig. 1). Our observations suggest that H. pylori induction of the NF-kB target gene TNFAIP3 can be advantageous for pathogen infection by specifically restricting host apoptotic cell death. This ‘pro-survival’ function of A20 can also be extrapolated to other pathogens, although different mechanisms are at work. In Tcells infected by human T-cell leukemia virus type I, A20 interacts directly with procaspase-8 and thus blocks the activation of procaspase-8 by preventing its recruitment to FADD. The parasite Leishmania donovani also benefits from infection-elicited NF-kB activation and the consecutive upregulation of A20. Here however, A20 functions in a different manner to support pathogen infection, namely by deubiquitinylation of TRAF6 to inhibit the Toll-like receptor 2 response and thereby circumvent the host immune response. For the intracellular pathogen Mycobacterium tuberculosis however, an effector molecule called ESAT-6 is secreted that downregulates miR-let-7f leading to the increase in expression of A20. Collectively, these findings increase our appreciation of what is likely hitherto an undervalued feature of infectioninduced NF-kB activity which plays to the benefit of the pathogen. With this recognition also comes the awareness that plausibly, with the multitude of host signaling pathways that are activated upon infection, there are still proteins to be discovered which have this ‘secondary action’ of enhancing the infection by pathogens. This should also be taken into consideration when evaluating the suitability of host proteins as therapeutic targets.