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Background: Acute myeloid leukemia (AML) is characterized by the abnormal proliferation of myeloid precursor cells and presents significant challenges in treatment due to its heterogeneity. Recently, the NLRP3 inflammasome has emerged as a potential contributor to AML pathogenesis, although its precise mechanisms remain poorly understood., Methods: Public genome datasets were utilized to evaluate the expression of NLRP3 inflammasome-related genes (IL-1β, IL-18, ASC, and NLRP3) in AML patients compared to healthy individuals. CRISPR/Cas9 technology was employed to generate NLRP3-deficient MOLM-13 AML cells, followed by comprehensive characterization using real-time PCR, western blotting, FACS analysis, and transmission electron and immunofluorescence microscopy. Proteomic analyses were conducted to identify NLRP3-dependent alterations in protein levels, with a focus on the eIF2 kinase PERK-mediated signaling pathways. Additionally, in vivo studies were performed using a leukemic mouse model to elucidate the pathogenic role of NLRP3 in AML., Results: Elevated expression of NLRP3 was significantly associated with diminished overall survival in AML patients. Genetic deletion, pharmacological inhibition and silencing by RNA interference of NLRP3 led to decreased AML cell survival through the induction of apoptosis. Proteomic analyses uncovered NLRP3-dependent alterations in protein translation, characterized by enhanced eIF2α phosphorylation in NLRP3-deficient AML cells. Moreover, inhibition of PERK-mediated eIF2α phosphorylation reduced apoptosis by downregulating pro-apoptotic Bcl-2 family members. In vivo studies demonstrated reduced leukemic burden in mice engrafted with NLRP3 knockout AML cells, as evidenced by alleviated leukemic symptoms., Conclusion: Our findings elucidate the involvement of the NLRP3/PERK/eIF2 axis as a novel driver of AML cell survival. Targeting NLRP3-induced signaling pathways, particularly through the PERK/eIF2 axis, presents a promising therapeutic strategy for AML intervention. These insights into the role of the NLRP3 inflammasome offer potential avenues for improving the prognosis and treatment outcomes of AML patients., (© 2024. The Author(s).)

Genetic variants in IL6ST encoding the shared cytokine receptor for the IL-6 cytokine family GP130 have been associated with a diverse number of clinical phenotypes and disorders. We provide a molecular classification for 59 reported rare IL6ST pathogenic or likely pathogenic variants and additional polymorphisms. Based on loss- or gain-of-function, cytokine selectivity, mono- and biallelic associations, and variable cellular mosaicism, we grade six classes of IL6ST variants and explore the potential for additional variants. We classify variants according to the American College of Medical Genetics and Genomics criteria. Loss-of-function variants with (i) biallelic complete loss of GP130 function that presents with extended Stüve-Wiedemann Syndrome; (ii) autosomal recessive hyper-IgE syndrome (HIES) caused by biallelic; and (iii) autosomal dominant HIES caused by monoallelic IL6ST variants both causing selective IL-6 and IL-11 cytokine loss-of-function defects; (iv) a biallelic cytokine-specific variant that exclusively impairs IL-11 signaling, associated with craniosynostosis and tooth abnormalities; (v) somatic monoallelic mosaic constitutively active gain-of-function variants in hepatocytes that present with inflammatory hepatocellular adenoma; and (vi) mosaic constitutively active gain-of-function variants in hematopoietic and non-hematopoietic cells that are associated with an immune dysregulation syndrome. In addition to Mendelian IL6ST coding variants, there are common non-coding cis-acting variants that modify gene expression, which are associated with an increased risk of complex immune-mediated disorders and trans-acting variants that affect GP130 protein function. Our taxonomy highlights IL6ST as a gene with particularly strong functional and phenotypic diversity due to the combinatorial biology of the IL-6 cytokine family and predicts additional genotype-phenotype associations., (© 2023. The Author(s).)

The cytokine interleukin-6 (IL-6) has considerable pro-inflammatory properties and is a driver of many physiological and pathophysiological processes. Cellular responses to IL-6 are mediated by membrane-bound or soluble forms of the IL-6 receptor (IL-6R) complexed with the signal-transducing subunit gp130. While expression of the membrane-bound IL-6R is restricted to selected cell types, soluble IL-6R (sIL-6R) enables gp130 engagement on all cells, a process termed IL-6 trans-signalling and considered to be pro-inflammatory. sIL-6R is predominantly generated through proteolytic processing by the metalloproteinase ADAM17. ADAM17 also liberates ligands of the epidermal growth factor receptor (EGFR), which is a prerequisite for EGFR activation and results in stimulation of proliferative signals. Hyperactivation of EGFR mostly due to activating mutations drives cancer development. Here, we reveal an important link between overshooting EGFR signalling and the IL-6 trans-signalling pathway. In epithelial cells, EGFR activity induces not only IL-6 expression but also the proteolytic release of sIL-6R from the cell membrane by increasing ADAM17 surface activity. We find that this derives from the transcriptional upregulation of iRhom2, a crucial regulator of ADAM17 trafficking and activation, upon EGFR engagement, which results in increased surface localization of ADAM17. Also, phosphorylation of the EGFR-downstream mediator ERK mediates ADAM17 activity via interaction with iRhom2. In sum, our study reveals an unforeseen interplay between EGFR activation and IL-6 trans-signalling, which has been shown to be fundamental in inflammation and cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

IL-6 plays a fundamental role in T cell differentiation and is strictly controlled by surface expression and shedding of IL-6R. IL-6 also acts on other cells that might affect T cell maturation. To study the impact of cell-autonomous and uncontrolled IL-6 signaling in T cells, we generated mice with a constitutively active IL-6R gp130 chain (Lgp130) expressed either in all T cells (Lgp130 × CD4Cre mice) or inducible in CD4+ T cells (Lgp130 × CD4CreERT2 mice). Lgp130 × CD4Cre mice accumulated activated T cells, including TH17 cells, in the lung, resulting in severe inflammation. Tamoxifen treatment of Lgp130 × CD4CreERT2 mice caused Lgp130 expression in 40-50% of CD4+ T cells, but mice developed lung disease only after several months. Lgp130+ CD4+ T cells were also enriched for TH17 cells; however, there was concomitant expansion of Lgp130- regulatory T cells, which likely restricted pathologic Lgp130+ T cells. In vitro, constitutive gp130 signaling in T cells enhanced but was not sufficient for TH17 cell differentiation. Augmented TH17 cell development of Lgp130+ T cells was also observed in Lgp130 × CD4CreERT2 mice infected with Staphylococcus aureus, but gp130 activation did not interfere with formation of TH1 cells against Listeria monocytogenes. Lgp130+ CD4+ T cells acquired a memory T cell phenotype and persisted in high numbers as a polyclonal T cell population in lymphoid and peripheral tissues, but we did not observe T cell lymphoma formation. In conclusion, cell-autonomous gp130 signaling alters T cell differentiation. Although gp130 signaling is not sufficient for TH17 cell differentiation, it still promotes accumulation of activated T cells in the lung that cause tissue inflammation., (Copyright © 2023 by The American Association of Immunologists, Inc.)

Background & Aims: Primary liver cancers include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (CCA) and combined HCC-CCA tumors (cHCC-CCA). It has been suggested, but not unequivocally proven, that hepatic progenitor cells (HPCs) can contribute to hepatocarcinogenesis. We aimed to determine whether HPCs contribute to HCC, cHCC-CCA or both types of tumors., Methods: To trace progenitor cells during hepatocarcinogenesis, we generated Mdr2-KO mice that harbor a yellow fluorescent protein (YFP) reporter gene driven by the Foxl1 promoter which is expressed specifically in progenitor cells. These mice (Mdr2-KO Foxl1-CRE;RosaYFP ) develop chronic inflammation and HCCs by the age of 14-16 months, followed by cHCC-CCA tumors at the age of 18 months., Results: In this Mdr2-KO Foxl1-CRE;RosaYFP mouse model, liver progenitor cells are the source of cHCC-CCA tumors, but not the source of HCC. Ablating the progenitors, caused reduction of cHCC-CCA tumors but did not affect HCCs. RNA-sequencing revealed enrichment of the IL-6 signaling pathway in cHCC-CCA tumors compared to HCC tumors. Single-cell RNA-sequencing (scRNA-seq) analysis revealed that IL-6 is expressed by immune and parenchymal cells during senescence, and that IL-6 is part of the senescence-associated secretory phenotype. Administration of an anti-IL-6 antibody to Mdr2-KO Foxl1-CRE;RosaYFP mice inhibited the development of cHCC-CCA tumors. Blocking IL-6 trans-signaling led to a decrease in the number and size of cHCC-CCA tumors, indicating their dependence on this pathway. Furthermore, the administration of a senolytic agent inhibited IL-6 and the development of cHCC-CCA tumors., Conclusion: Our results demonstrate that cHCC-CCA, but not HCC tumors, originate from HPCs, and that IL-6, which derives in part from cells in senescence, plays an important role in this process via IL-6 trans-signaling. These findings could be applied to develop new therapeutic approaches for cHCC-CCA tumors., Lay Summary: Combined hepatocellular carcinoma-cholangiocarcinoma is the third most prevalent type of primary liver cancer (i.e. a cancer that originates in the liver). Herein, we show that this type of cancer originates in stem cells in the liver and that it depends on inflammatory signaling. Specifically, we identify a cytokine called IL-6 that appears to be important in the development of these tumors. Our results could be used for the development of novel treatments for these aggressive tumors., Competing Interests: Conflict of interest S.R.-J. is an inventor on patents owned by CONARIS Research Institute, which develops the sgp130Fc protein, and he has stock ownership in CONARIS. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Ectodomain shedding is an irreversible process to regulate inter- and intracellular signaling. Members of the a disintegrin and metalloprotease (ADAM) family are major mediators of ectodomain shedding. ADAM17 is involved in the processing of multiple substrates including tumor necrosis factor (TNF) α and EGF receptor ligands. Substrates of ADAM17 are selectively processed depending on stimulus and cellular context. However, it still remains largely elusive how substrate selectivity of ADAM17 is regulated. Tetraspanins (Tspan) are multi-membrane-passing proteins that are involved in the organization of plasma membrane micro-domains and diverse biological processes. Closely related members of the Tspan8 subfamily, including CD9, CD81 and Tspan8, are associated with cancer and metastasis. Here, we show that Tspan8 subfamily members use different strategies to regulate ADAM17 substrate selectivity. We demonstrate that in particular Tspan8 associates with both ADAM17 and TNF α and promotes ADAM17-mediated TNF α release through recruitment of ADAM17 into Tspan-enriched micro-domains. Yet, processing of other ADAM17 substrates is not altered by Tspan8. We, therefore, propose that Tspan8 contributes to tumorigenesis through enhanced ADAM17-mediated TNF α release and a resulting increase in tissue inflammation.

Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies., Competing Interests: Disclosures: I. Sagi reported a patent (no. US 10,933,122 B2) issued. D. Schmidt-Arras reported personal fees from Mestag Therapeutics Ltd. outside the submitted work. No other disclosures were reported., (© 2021 Bolik et al.)

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.

Background: Biallelic variants in IL6ST, encoding GP130, cause a recessive form of hyper-IgE syndrome (HIES) characterized by high IgE level, eosinophilia, defective acute phase response, susceptibility to bacterial infections, and skeletal abnormalities due to cytokine-selective loss of function in GP130, with defective IL-6 and IL-11 and variable oncostatin M (OSM) and IL-27 levels but sparing leukemia inhibitory factor (LIF) signaling., Objective: Our aim was to understand the functional and structural impact of recessive HIES-associated IL6ST variants., Methods: We investigated a patient with HIES by using exome, genome, and RNA sequencing. Functional assays assessed IL-6, IL-11, IL-27, OSM, LIF, CT-1, CLC, and CNTF signaling. Molecular dynamics simulations and structural modeling of GP130 cytokine receptor complexes were performed., Results: We identified a patient with compound heterozygous novel missense variants in IL6ST (p.Ala517Pro and the exon-skipping null variant p.Gly484_Pro518delinsArg). The p.Ala517Pro variant resulted in a more profound IL-6- and IL-11-dominated signaling defect than did the previously identified recessive HIES IL6ST variants p.Asn404Tyr and p.Pro498Leu. Molecular dynamics simulations suggested that the p.Ala517Pro and p.Asn404Tyr variants result in increased flexibility of the extracellular membrane-proximal domains of GP130. We propose a structural model that explains the cytokine selectivity of pathogenic IL6ST variants that result in recessive HIES. The variants destabilized the conformation of the hexameric cytokine receptor complexes, whereas the trimeric LIF-GP130-LIFR complex remained stable through an additional membrane-proximal interaction. Deletion of this membrane-proximal interaction site in GP130 consequently caused additional defective LIF signaling and Stüve-Wiedemann syndrome., Conclusion: Our data provide a structural basis to understand clinical phenotypes in patients with IL6ST variants., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The liver is a vital organ with multiple functions and a large regenerative capacity. Tumours of the liver are the second most frequently cause of cancer-related death and develop in chronically inflamed livers. IL-6-type cytokines are mediators of inflammation and almost all members signal via the receptor subunit gp130 and the downstream signalling molecule STAT3. We here summarize current knowledge on how gp130 signalling and STAT3 in tumour cells and cells of the tumour micro-environment drives hepatic tumorigenesis. We furthermore discuss very recent findings describing also anti-tumorigenic roles of gp130/STAT3 and important considerations for therapeutic interventions., (© 2021. The Author(s).)

Interleukin-6 (IL-6) is the name-giving cytokine of a family of eleven members, including IL-6, CNTF, LIF, and IL-27. IL-6 was first recognized as a B-cell stimulating factor but we now know that the cytokine plays a pivotal role in the orchestration of inflammatory processes as well as in inflammation associated cancer. Moreover, IL-6 is involved in metabolic regulation and it has been shown to be involved in major neural activities such as neuroprotection, which can help to repair and to reduce brain damage. Receptor complexes of all members formed at the plasma membrane contain one or two molecules of the signaling receptor subunit GP130 and the mechanisms of signal transduction are well understood. IL-6 type cytokines can also signal from endomembranes, in particular the endosome, and situations have been reported in which endocytosis of receptor complexes are a prerequisite of intracellular signaling. Moreover, pathogenic GP130 variants were shown to interfere with spatial activation of downstream signals. We here summarize the molecular mechanisms underlying spatial regulation of IL-6 family cytokine signaling and discuss its relevance for pathogenic processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schmidt-Arras and Rose-John.)

Background & Aims: Interleukin (IL)-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signaling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6-type cytokines, making it difficult to delineate cell type-specific effects. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signaling., Methods: We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signaling in distinct liver cell types: hepatic stellate cells, cholangiocytes/liver progenitor cells or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model., Results: Hepatocyte-specific gp130 activation led to the upregulation of innate immune system components, including acute-phase proteins. Consequently, we observed peripheral mobilization and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of hepatic stellate cell- or cholangiocyte/liver progenitor cell-specific transgenic gp130 signaling., Conclusions: Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NF-κB activation. We therefore conclude that gp130 engagement, e.g. by IL-6 trans-signaling, represents a safe-guard mechanism in innate immunity., Lay Summary: Members of the interleukin-6 cytokine family signal via the receptor subunit gp130 and are involved in multiple processes in the liver. However, as several liver cell types respond to interleukin-6 family cytokines, it is difficult to delineate cell type-specific effects. Using a novel mouse model, we provide evidence that hepatocyte-specific gp130 activation is sufficient to trigger a robust systemic innate immune response., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Fms-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinases (RTK) and is involved in cell survival, proliferation, and differentiation of haematopoietic progenitors of lymphoid and myeloid lineages. Oncogenic mutations in the FLT3 gene resulting in constitutively active FLT3 variants are frequently found in acute myeloid leukaemia (AML) patients and correlate with patient's poor survival. Targeting FLT3 mutant leukaemic stem cells (LSC) is a key to efficient treatment of patients with relapsed/refractory AML. It is therefore essential to understand how LSC escape current therapies in order to develop novel therapeutic strategies. Here, we summarize the current knowledge on mechanisms of FLT3 activity regulation and its cellular consequences. Furthermore, we discuss how aberrant FLT3 signalling cooperates with other oncogenic lesions and the microenvironment to drive haematopoietic malignancies and how this can be harnessed for therapeutical purposes.

Activating mutations in genes encoding receptor tyrosine kinases (RTKs) mediate proliferation, cell migration, and cell survival, and are therefore important drivers of oncogenesis. Numerous targeted cancer therapies are directed against activated RTKs, including small compound inhibitors, and immunotherapies. It has recently been discovered that not only certain RTK fusion proteins, but also many full-length RTKs harbouring activating mutations, notably RTKs of the class III family, are to a large extent mislocalised in intracellular membranes. Active kinases in these locations cause aberrant activation of signalling pathways. Moreover, low levels of activated RTKs at the cell surface present an obstacle for immunotherapy. We outline here why understanding of the mechanisms underlying mislocalisation will help in improving existing and developing novel therapeutic strategies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Tumour growth is not solely driven by tumour cell-intrinsic mechanisms, but also depends on paracrine signals provided by the tumour micro-environment. These signals comprise cytokines and growth factors that are synthesized as trans-membrane proteins and need to be liberated by limited proteolysis also termed ectodomain shedding. Members of the family of A disintegrin and metalloproteases (ADAM) are major mediators of ectodomain shedding and therefore initiators of paracrine signal transduction. In this review, we summarize the current knowledge on how ADAM proteases on tumour cells but also on cells of the tumour micro-environment contribute to the formation of gastrointestinal tumours, and discuss how these processes can be exploited pharmacologically.

The GP130 cytokine receptor subunit encoded by IL6ST is the shared receptor for ten cytokines of the IL-6 family. We describe a homozygous non-synonymous variant in IL6ST (p.R281Q) in a patient with craniosynostosis and retained deciduous teeth. We characterize the impact of the variant on cytokine signaling in vitro using transfected cell lines as well as primary patient-derived cells and support these findings using a mouse model with the corresponding genome-edited variant Il6st p.R279Q. We show that human GP130 p.R281Q is associated with selective loss of IL-11 signaling without affecting IL-6, IL-27, OSM, LIF, CT1, CLC, and CNTF signaling. In mice Il6st p.R279Q lowers litter size and causes facial synostosis and teeth abnormalities. The effect on IL-11 signaling caused by the GP130 variant shows incomplete penetrance but phenocopies aspects of IL11RA deficiency in humans and mice. Our data show that a genetic variant in a pleiotropic cytokine receptor can have remarkably selective defects., Competing Interests: Competing interestsNo conflict of interest related to this article. H.H.U. has received research support or consultancy fees from UCB Pharma, Eli Lilly, Boehringer Ingelheim, Pfizer, Celgene, MiroBio and AbbVie. H.H.U., A.L., and Y.-H.C are supported by a research collaboration with Celgene., (© The Author(s) 2020.)

Objective: Failing to properly repair damaged DNA drives the ageing process. Furthermore, age-related inflammation contributes to the manifestation of ageing. Recently, we demonstrated that the efficiency of repair of diethylnitrosamine (DEN)-induced double-strand breaks (DSBs) rapidly declines with age. We therefore hypothesised that with age, the decline in DNA damage repair stems from age-related inflammation., Design: We used DEN-induced DNA damage in mouse livers and compared the efficiency of their resolution in different ages and following various permutations aimed at manipulating the liver age-related inflammation., Results: We found that age-related deregulation of innate immunity was linked to altered gut microbiota. Consequently, antibiotic treatment, MyD88 ablation or germ-free mice had reduced cytokine expression and improved DSBs rejoining in 6-month-old mice. In contrast, feeding young mice with a high-fat diet enhanced inflammation and facilitated the decline in DSBs repair. This latter effect was reversed by antibiotic treatment. Kupffer cell replenishment or their inactivation with gadolinium chloride reduced proinflammatory cytokine expression and reversed the decline in DSBs repair. The addition of proinflammatory cytokines ablated DSBs rejoining mediated by macrophage-derived heparin-binding epidermal growth factor-like growth factor., Conclusions: Taken together, our results reveal a previously unrecognised link between commensal bacteria-induced inflammation that results in age-dependent decline in DNA damage repair. Importantly, the present study support the notion of a cell non-autonomous mechanism for age-related decline in DNA damage repair that is based on the presence of 'inflamm-ageing' cytokines in the tissue microenvironment, rather than an intrinsic cellular deficiency in the DNA repair machinery., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Disintegrin and metalloproteinases (ADAMs) 10 and 17 can release the extracellular part of a variety of membrane-bound proteins via ectodomain shedding important for many biological functions. So far, substrate identification focused exclusively on membrane-anchored ADAM10 and ADAM17. However, besides known shedding of ADAM10, we identified ADAM8 as a protease capable of releasing the ADAM17 ectodomain. Therefore, we investigated whether the soluble ectodomains of ADAM10/17 (sADAM10/17) exhibit an altered substrate spectrum compared to their membrane-bound counterparts. A mass spectrometry-based N-terminomics approach identified 134 protein cleavage events in total and 45 common substrates for sADAM10/17 within the secretome of murine cardiomyocytes. Analysis of these cleavage sites confirmed previously identified amino acid preferences. Further in vitro studies verified fibronectin, cystatin C, sN-cadherin, PCPE-1 as well as sAPP as direct substrates of sADAM10 and/or sADAM17. Overall, we present the first degradome study for sADAM10/17, thereby introducing a new mode of proteolytic activity within the protease web.

Fibrosis in the liver is mainly associated with the activation of hepatic stellate cells (HSCs). Both activation and clearance of HSCs can be mediated by ligand-receptor interactions. Members of the a disintegrin and metalloprotease (ADAM) family are involved in the proteolytic release of membrane-bound ligands and receptor ectodomains and the remodelling of the extracellular matrix. ADAM proteases are therefore major regulators of intercellular signalling pathways. In the present review we discuss how ADAM proteases modulate pro- and anti-fibrotic processes and how ADAM proteases might be harnessed therapeutically in the future.

Multi drug resistance protein 2 knockout mice (Mdr2 -/- ) are a mouse model of chronic liver inflammation and inflammation-induced tumour development. Here we investigated the kinetics of early heme oxygenase 1 (HO-1) induction on inflammation, tumour development, and DNA damage in Mdr2 -/- mice. HO-1 was induced by intraperitoneal injection of cobalt protoporphyrin IX (CoPP) twice weekly for 9 consecutive weeks. Immediately after HO-1 induction, liver function improved and infiltration of CD4 + and CD8 + T cells was reduced. Furthermore, we observed increased p38 activation with concomitant reduction of Cyclin D1 expression in aged Mdr2 -/- mice. Long-term effects of HO-1 induction included increased CD8 + T cell infiltration as well as delayed and reduced tumour growth in one-year-old animals. Unexpectedly, DNA double-strand breaks were detected predominantly in macrophages of 65-week-old Mdr2 -/- mice, while DNA damage was reduced in response to early HO-1 induction in vivo and in vitro. Overall, early induction of HO-1 in Mdr2 -/- mice had a beneficial short-term effect on liver function and reduced hepatic T cell accumulation. Long-term effects of early HO-1 induction were increased CD8 + T cell numbers, decreased proliferation as wells as reduced DNA damage in liver macrophages of aged animals, accompanied by delayed and reduced tumour growth.

Colorectal cancer is treated with antibodies blocking epidermal growth factor receptor (EGF-R), but therapeutic success is limited. EGF-R is stimulated by soluble ligands, which are derived from transmembrane precursors by ADAM17-mediated proteolytic cleavage. In mouse intestinal cancer models in the absence of ADAM17, tumorigenesis was almost completely inhibited, and the few remaining tumors were of low-grade dysplasia. RNA sequencing analysis demonstrated down-regulation of STAT3 and Wnt pathway components. Because EGF-R on myeloid cells, but not on intestinal epithelial cells, is required for intestinal cancer and because IL-6 is induced via EGF-R stimulation, we analyzed the role of IL-6 signaling. Tumor formation was equally impaired in IL-6 -/- mice and sgp130Fc transgenic mice, in which only trans-signaling via soluble IL-6R is abrogated. ADAM17 is needed for EGF-R-mediated induction of IL-6 synthesis, which via IL-6 trans-signaling induces β-catenin-dependent tumorigenesis. Our data reveal the possibility of a novel strategy for treatment of colorectal cancer that could circumvent intrinsic and acquired resistance to EGF-R blockade., (© 2018 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.)

Chemotherapeutic resistance evolves in about 70 % of ovarian cancer patients and is a major cause of death in this tumor entity. Novel approaches to overcome these therapeutic limitations are therefore highly warranted. A disintegrin and metalloprotease 17 (ADAM17) is highly expressed in ovarian cancer and required for releasing epidermal growth factor receptor (EGFR) ligands like amphiregulin (AREG). This factor has recently been detected in ascites of advanced stage ovarian cancer patients. However, it is not well understood, whether and how ADAM17 might contribute to chemo resistance of ovarian cancer. In this study, we identified ADAM17 as an essential upstream regulator of AREG release under chemotherapeutic treatment in ovarian cancer cell lines and patient derived cells. In the majority of ovarian cancer cells cisplatin treatment resulted in enhanced ADAM17 activity, as shown by an increased shedding of AREG. Moreover, both mRNA and the protein content of AREG were dose-dependently increased by cisplatin exposure. Consequently, cisplatin strongly induced phosphorylation of ADAM17-downstream mediators, the EGFR and extracellular signal-regulated kinases (ERK). Phorbol 12-myristate 13-acetate (PMA), similarly to cisplatin, mediated AREG shedding and membrane fading of surface ADAM17. Inhibition of ADAM17 with either GW280264X or the anti-ADAM17 antibody D1 (A12) as well as silencing of ADAM17 by siRNA selectively reduced AREG release. Thus, ADAM17 inhibition sensitized cancer cells to cisplatin-induced apoptosis, and significantly reduced cell viability. Based on these findings, we propose that targeting of ADAM17 in parallel to chemotherapeutic treatment suppresses survival pathways and potentially diminish evolving secondary chemo resistance mechanisms., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflict of interest.

The disintegrin metalloprotease ADAM17 has a critical role in intestinal inflammation and regeneration in mice, as illustrated by the dramatically increased susceptibility of ADAM17 hypomorphic (ADAM17 ex/ex ) mice to dextran sulfate sodium (DSS)-induced colitis. Similarly, necroptosis has been implicated in inflammatory responses in the intestine. In this study, we have investigated the contribution of necroptosis to ADAM17-regulated intestinal inflammation in vivo by crossing ADAM17 ex/ex mice with mice that lack the necroptotic core protein RIPK3. Despite the loss of RIPK3, ADAM17 ex/ex /RIPK3 -/- mice showed the same increased susceptibility as ADAM17 ex/ex mice in both acute and chronic models of DSS-induced colitis. Mice of both genotypes revealed comparable results with regard to weight loss, disease activity index and colitis-associated changes of inner organs. Histopathological analyses confirmed similar tissue destruction, loss of barrier integrity, immune cell infiltration, and cell death; serum analyses revealed similar levels of the pro-inflammatory cytokine KC. Resolving these unexpected findings, ADAM17 ex/ex mice did not show phosphorylation of RIPK3 and its necroptotic interaction partner MLKL during DSS-induced colitis, although both proteins were clearly expressed. Consistent with these findings, murine embryonic fibroblasts derived from ADAM17 ex/ex mice were protected from tumor necrosis factor (TNF)-induced necroptosis and failed to show phosphorylation of MLKL and RIPK3 after induction of necroptosis by TNF, revealing a novel, undescribed role of the protease ADAM17 in necroptosis., Competing Interests: CONFLICTS OF INTEREST The authors of this manuscript state that they do not have any financial conflict of interests and nothing to disclose. All authors have approved the manuscript.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

Competing Interests: Competing interests: None declared.

Multiple cytokines, including interleukin 6 (IL-6), IL-11, IL-27, oncostatin M (OSM), and leukemia inhibitory factor (LIF), signal via the common GP130 cytokine receptor subunit. In this study, we describe a patient with a homozygous mutation of IL6ST (encoding GP130 p.N404Y) who presented with recurrent infections, eczema, bronchiectasis, high IgE, eosinophilia, defective B cell memory, and an impaired acute-phase response, as well as skeletal abnormalities including craniosynostosis. The p.N404Y missense substitution is associated with loss of IL-6, IL-11, IL-27, and OSM signaling but a largely intact LIF response. This study identifies a novel immunodeficiency with phenotypic similarities to STAT3 hyper-IgE syndrome caused by loss of function of GP130., (© 2017 Schwerd et al.)

Fms-like tyrosine kinase 3 (FLT3) with internal tandem duplications (ITD) is a major oncoprotein in acute myeloid leukemia (AML), and confers an unfavorable prognosis. Interference with FLT3ITD signaling is therefore pursued as a promising therapeutic strategy. In this study we show that abrogation of FLT3ITD glycoprotein maturation using low doses of the N-glycosylation inhibitor tunicamycin has anti-proliferative and pro-apoptotic effects on FLT3ITD-expressing human and murine cell lines. This effect is mediated in part by arresting FLT3ITD in an underglycosylated state and thereby attenuating FLT3ITD-driven AKT and ERK signaling. In addition, tunicamycin caused pronounced endoplasmatic reticulum stress and apoptosis through activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activation of the gene encoding CCAAT-enhancer-binding protein homologous protein (CHOP). PERK inhibition with a small molecule attenuated CHOP induction and partially rescued cells from apoptosis. Combination of tunicamycin with potent FLT3ITD kinase inhibitors caused synergistic cell killing, which was highly selective for cell lines and primary AML cells expressing FLT3ITD. Although tunicamycin is currently not a clinically applicable drug, we propose that mild inhibition of N-glycosylation may have therapeutic potential in combination with FLT3 kinase inhibitors for FLT3ITD-positive AML.

Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide with rising incidence. The inflammatory cytokine, interleukin-6 (IL-6), is a critical mediator of HCC development. It can signal through two distinct pathways: the IL-6 classic and the IL-6 trans-signaling pathway. Whereas IL-6 classic signaling is important for innate and acquired immunity, IL-6 trans-signaling has been linked to accelerated liver regeneration and several chronic inflammatory pathologies. However, its implication in liver tumorigenesis has not been addressed yet. Here, we show that IL-6 trans-signaling, but not IL-6 classic signaling, is essential to promote hepatocellular carcinogenesis by two mechanisms: First, it prevents DNA-damage-induced hepatocyte apoptosis through suppression of p53 and enhances β-catenin activation and tumor proliferation. Second, IL-6 trans-signaling directly induces endothelial cell proliferation to promote tumor angiogenesis. Consequently, soluble gp130 fused to Fc transgenic mice lacking IL-6 trans-signaling are largely protected from tumor formation in a diethylnitrosamine/3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene model of HCC., Conclusion: IL-6 trans-signaling, and not IL-6 classic signaling, is mandatory for development of hepatocellular carcinogenesis. Therefore, specific inhibition of IL-6 trans-signaling, rather than total inhibition of IL-6 signaling, is sufficient to blunt tumor initiation and impair tumor progression without compromising IL-6 classic signaling-driven protective immune responses. (Hepatology 2017;65:89-103)., (© 2016 by the American Association for the Study of Liver Diseases.)

Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified.

Pro-inflammatory signaling pathways, especially interleukin 6 (IL-6), and reactive oxygen species (ROS) promote carcinogenesis in the liver. In order to elucidate the underlying oncogenic mechanism, we activated the IL-6 signal transducer glycoprotein 130 (gp130) via stable expression of a constitutively active gp130 construct (L-gp130) in untransformed telomerase-immortalized human fetal hepatocytes (FH-hTERT). As known from hepatocellular adenomas, forced gp130 activation alone was not sufficient to induce malignant transformation. However, additional challenge of FH-hTERT L-gp130 clones with oxidative stress resulted in 2- to 3-fold higher ROS levels and up to 6-fold more DNA-double strand breaks (DSB). Despite increased DNA damage, ROS-challenged FH-hTERT L-gp130 clones displayed an enhanced proliferation and rapidly developed colony growth capabilities in soft agar. As driving gp130-mediated oncogenic mechanism, we detected a decreased expression of antioxidant genes, in particular glutathione peroxidase 3 and apolipoprotein E, and an absence of P21 upregulation following ROS-conferred induction of DSB. In summary, an impaired oxidative stress response in hepatocytes with gp130 gain-of-function mutations, as detected in dysplastic intrahepatic nodules and hepatocellular adenomas, is one of the central oncogenic mechanisms in chronic liver inflammation.

Interleukin 6 (IL-6) is a pleiotropic four-helix-bundle cytokine that exerts multiple functions in the body. In the liver, IL-6 is an important inducer of the acute phase response and infection defense. IL-6 is furthermore crucial for hepatocyte homeostasis and is a potent hepatocyte mitogen. It is not only implicated in liver regeneration, but also in metabolic function of the liver. However, persistent activation of the IL-6 signaling pathway is detrimental to the liver and might ultimately result in the development of liver tumors. On target cells IL-6 can bind to the signal transducing subunit gp130 either in complex with the membrane-bound or with the soluble IL-6 receptor to induce intracellular signaling. In this review we describe how these different pathways are involved in the physiology and pathophyiology of the liver. We furthermore discuss how IL-6 pathways can be selectively inhibited and therapeutically exploited for the treatment of liver pathologies., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Unlabelled: A Disintegrin And Metalloprotease (ADAM) 10 exerts essential roles during organ development and tissue integrity in different organs, mainly through activation of the Notch pathway. However, only little is known about its implication in liver tissue physiology. Here we show that in contrast to its role in other tissues, ADAM10 is dispensable for the Notch2-dependent biliary tree formation. However, we demonstrate that expression of bile acid transporters is dependent on ADAM10. Consequently, mice deficient for Adam10 in hepatocytes, cholangiocytes and liver progenitor cells develop spontaneous hepatocyte necrosis and concomitant liver fibrosis. We furthermore observed a strongly augmented ductular reaction in 15-week old ADAM10(Δhep/Δch) mice and demonstrate that c-Met dependent liver progenitor cell activation is enhanced. Additionally, liver progenitor cells are primed to hepatocyte differentiation in the absence of ADAM10. These findings show that ADAM10 is a novel central node controlling liver tissue homeostasis., Highlights: Loss of ADAM10 in murine liver results in hepatocyte necrosis and concomitant liver fibrosis. ADAM10 directly regulates expression of bile acid transporters but is dispensable for Notch2-dependent formation of the biliary system. Activation of liver progenitor cells is enhanced through increased c-Met signalling, in the absence of ADAM10. Differentiation of liver progenitor cells to hepatocytes is augmented in the absence of ADAM10.

Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage-specifically regulated, as its kinase activity increases during the promastigote to amastigote conversion. However, unlike canonical MAPKs that are activated by dual phosphorylation of the regulatory TxY motif in the activation loop, MPK10 activation is independent from the phosphorylation of the tyrosine residue, which is largely constitutive. Removal of the last 46 amino acids resulted in significantly enhanced MPK10 activity both for the recombinant and transgenic protein, revealing that MPK10 is regulated by an auto-inhibitory mechanism. Over-expression of this hyperactive mutant in transgenic parasites led to a dominant negative effect causing massive cell death during amastigote differentiation, demonstrating the essential nature of MPK10 auto-inhibition for parasite viability. Moreover, phosphoproteomics analyses identified a novel regulatory phospho-serine residue in the C-terminal auto-inhibitory domain at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10 as a potential signal sensor of the mammalian host environment, whose intrinsic pre-activated conformation is regulated by auto-inhibition.

ADAM17 (a disintegrin and metalloprotease 17) controls pro- and anti-inflammatory signaling events by promoting ectodomain shedding of cytokine precursors and cytokine receptors. Despite the well documented substrate repertoire of ADAM17, little is known about regulatory mechanisms, leading to substrate recognition and catalytic activation. Here we report a direct interaction of the acidophilic kinase Polo-like kinase 2 (PLK2, also known as SNK) with the cytoplasmic portion of ADAM17 through the C-terminal noncatalytic region of PLK2 containing the Polo box domains. PLK2 activity leads to ADAM17 phosphorylation at serine 794, which represents a novel phosphorylation site. Activation of ADAM17 by PLK2 results in the release of pro-TNFα and TNF receptors from the cell surface, and pharmacological inhibition of PLK2 leads to down-regulation of LPS-induced ADAM17-mediated shedding on primary macrophages and dendritic cells. Importantly, PLK2 expression is up-regulated during inflammatory conditions increasing ADAM17-mediated proteolytic events. Our findings suggest a new role for PLK2 in the regulation of inflammatory diseases by modulating ADAM17 activity.

Interleukin 6 (IL-6) and, hence, activation of the IL-6 receptor signalling subunit glycoprotein 130 (gp130; also known as interleukin-6 receptor subunit β, IL6ST), has been linked to inflammation and tumour formation. Recently, deletion mutations in gp130 have been identified in inflammatory hepatocellular adenoma. The mutations clustered around one IL-6-binding epitope and rendered gp130 constitutively active in a ligand-independent manner. Here, we show that gp130 deletion mutants, but not wild-type gp130, localise predominantly to intracellular compartments, notably the endoplasmic reticulum (ER) and early endosomes. One of the most frequent mutants, gp130 Y186-Y190del (ΔYY) is retained in the ER quality control system because of its association with the chaperone calnexin. Furthermore, we can show that gp130 ΔYY induces downstream signalling from both ER and endosomes, and that both signals contribute to ligand-independent cell proliferation. We also demonstrate that the endosomal localisation of gp130 ΔYY is crucial for fully fledged STAT3 activation. Therefore, aberrant signalling from intracellular compartments might explain the tumorigenic potential of naturally occurring somatic mutations of gp130.