Dirk Schmidt-Arras

The pro- and anti-inflammatory properties of the cytokine interleukin-6.

Interleukin-6 is a cytokine not only involved in inflammation and infection responses but also in the regulation of metabolic, regenerative, and neural processes. In classic signaling, interleukin-6 stimulates target cells via a membrane bound interleukin-6 receptor, which upon ligand binding associates with the signaling receptor protein gp130. Gp130 dimerizes, leading to the activation of Janus kinases and subsequent phosphorylation of tyrosine residues within the cytoplasmic portion of gp130. This leads to the engagement of phosphatase Src homology domains containing tyrosin phosphatase-2 (SHP-2) and activation of the ras/raf/Mitogen-activated protein (MAP) kinase (MAPK) pathway. In addition, signal transducer and activator of transcription factors are recruited, which are phosphorylated, and consequently dimerize whereupon they translocate into the nucleus and activate target genes. Interestingly, only few cells express membrane bound interleukin-6 receptor whereas all cells display gp130 on the cell surface. While cells, which only express gp130, are not responsive to interleukin-6 alone, they can respond to a complex of interleukin-6 bound to a naturally occurring soluble form of the interleukin-6 receptor. Therefore, the generation of soluble form of the interleukin-6 receptor dramatically enlarges the spectrum of interleukin-6 target cells. This process has been named trans-signaling. Here, we review the involvement of both signaling modes in the biology of interleukin-6. It turns out that regenerative or anti-inflammatory activities of interleukin-6 are mediated by classic signaling whereas pro-inflammatory responses of interleukin-6 are rather mediated by trans-signaling. This is important since therapeutic blockade of interleukin-6 by the neutralizing anti-interleukin-6 receptor monoclonal antibody tocilizumab has recently been approved for the treatment of inflammatory diseases. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

IL-6 pathway in the liver: From physiopathology to therapy

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.

Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10

A disintegrin and metalloproteinase10 (ADAM10) has been implicated as a major sheddase responsible for the ectodomain shedding of a number of important surface molecules including the amyloid precursor protein and cadherins. Despite a well-documented role of ADAM10 in health and disease, little is known about the regulation of this protease. To address this issue we conducted a split-ubiquitin yeast two-hybrid screen to identify membrane proteins that interact with ADAM10. The yeast experiments and co-immunoprecipitation studies in mammalian cell lines revealed tetraspanin15 (TSPAN15) to specifically associate with ADAM10. Overexpression of TSPAN15 or RNAi-mediated knockdown of TSPAN15 led to significant changes in the maturation process and surface expression of ADAM10. Expression of an endoplasmic reticulum (ER) retention mutant of TSPAN15 demonstrated an interaction with ADAM10 already in the ER. Pulse-chase experiments confirmed that TSPAN15 accelerates the ER-exit of the ADAM10–TSPAN15 complex and stabilizes the active form of ADAM10 at the cell surface. Importantly, TSPAN15 also showed the ability to mediate the regulation of ADAM10 protease activity exemplified by an increased shedding of N-cadherin and the amyloid precursor protein. In conclusion, our data show that TSPAN15 is a central modulator of ADAM10-mediated ectodomain shedding. Therapeutic manipulation of its expression levels may be an additional approach to specifically regulate the activity of the amyloid precursor protein alpha-secretase ADAM10.

Flt3 receptor tyrosine kinase as a drug target in leukemia.

The hematopoietic class III receptor tyrosine kinase (RTK) Flt3 (Flk2, STK1) has recently received much attention as a potential drug target. Activation of Flt3 by different types of mutations plays an important role for proliferation, resistance to apoptosis, and prevention of differentiation of leukemic blasts in acute myeloid leukemia (AML). At least one type of such mutations - an internal tandem duplication in the Flt3 juxtamembrane domain (Flt3-ITD) - has been associated with an unfavorable prognosis. Signal transduction of Flt3 involves activation of several conserved pathways, including the RAS/MAP-Kinase and the phosphoinositide-3-kinase/Akt signaling cascades. Transforming versions of Flt3 exhibit altered signaling, for example a very pronounced activation of STAT5, ultimately resulting in alternate profiles of gene expression and cell transformation. Selective inhibitors of Flt3 tyrosine kinase activity have the potential to suppress aberrant Flt3 signaling. Although highly homologous to other class III RTKs, Flt3 is resistant to the phenylaminopyrimidine STI571 (Gleevec, Imatinib), a potent inhibitor of other RTKs in the family, such as the PDGFbeta-receptor or c-Kit. STI571 binding to Flt3 is prevented by the phenylalanine 691 side-chain in the ATP binding center and mutating this site to threonine renders the corresponding Flt3 mutant sensitive to STI571. Compounds of several other structural families, including the quinoxaline AG1296, the bis(1H-2-indolyl)-1-methanone D-65476, the indolinones SU5416 and SU11248, the indolocarbazoles PKC412 and CEP-701, and the piperazonyl quinazoline CT53518, are potent inhibitors of Flt3 kinase. They exhibit different selectivity profiles, both with respect to other kinases and among wildtype Flt3 and its activated versions. These compounds hold promise as novel drugs against AML and as probes for understanding activation mechanisms and signaling pathways in the class III RTK family.

Anchoring of FLT3 in the endoplasmic reticulum alters signaling quality

The mechanism of cell transformation by Fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is incompletely understood. The most prevalent activated mutant FLT3 ITD exhibits an altered signaling quality, including strong activation of the STAT5 transcription factor. FLT3 ITD has also been found partially retained as a high-mannose precursor in an intracellular compartment. To analyze the role of intracellular retention of FLT3 for transformation, we have generated FLT3 versions that are anchored in the perinuclear endoplasmic reticulum (ER) by appending an ER retention sequence containing a RRR (R3) motif. ER retention of R3, but not of corresponding A3 FLT3 versions, is shown by biochemical, fluorescence-activated cell sorting, and immunocytochemical analyses. ER anchoring reduced global autophosphorylation and diminished constitutive activation of ERK1/2 and AKT of the constitutively active FLT3 versions. ER anchoring was, however, associated with elevated signaling to STAT3. Transforming activity of the FLT3 D835Y mutant was suppressed by ER anchoring. In contrast, ER-anchored FLT3 ITD retained STAT5-activating capacity and was transforming in vitro and in vivo. The findings highlight another aspect of the different signaling quality of FLT3 ITD: It can transform cells from an intracellular location.

Cellular Inhibition of Protein Tyrosine Phosphatase 1B by Uncharged Thioxothiazolidinone Derivatives

As important regulators of cellular signal transduction, members of the protein tyrosine phosphatase (PTP) family are considered to be promising drug targets. However, to date, the most effective in vitro PTP inhibitors have tended to be highly charged, thus limiting cellular permeability. Here, we have identified an uncharged thioxothiazolidinone derivative (compound 1), as a competitive inhibitor of a subset of PTPs. Compound 1 effectively inhibited protein tyrosine phosphatase 1B (PTP1B) in two cell‐based systems: it sensitized wild‐type, but not PTP1B‐null fibroblasts to insulin stimulation and prevented PTP1B‐dependent dephosphorylation of the FLT3‐ITD receptor tyrosine kinase. We have also tested a series of derivatives in vitro against PTP1B and proposed a model of the PTP1B–inhibitor interaction. These compounds should be useful in the elucidation of cellular PTP function and could represent a starting point for development of therapeutic PTP inhibitors.

Hepatocytes Contribute to Immune Regulation in the Liver by Activation of the Notch Signaling Pathway in T Cells

The “liver tolerance effect” has been attributed to a unique potential of liver-resident nonprofessional APCs including hepatocytes (HCs) to suppress T cell responses. The exact molecular mechanism of T cell suppression by liver APCs is still largely unknown. In mice, IL-10–dependent T cell suppression is observed after Th1-mediated hepatitis induced by Con A. In this study, we show that HCs, particularly those from regenerating livers of Con A–pretreated mice, induced a regulatory phenotype in naive CD4+ T cells in vitro. Using reporter mice, we observed that these T regulatory cells released substantial amounts of IL-10, produced IFN-γ, failed to express Foxp3, but suppressed proliferation of responder T cells upon restimulation with anti-CD3 mAb. Hence, these regulatory cells feature a similar phenotype as the recently described IL-10–producing Th1 cells, which are generated upon activation of Notch signaling. Indeed, inhibition of γ-secretase and a disintegrin and metalloproteinase 17 but not a disintegrin and metalloproteinase 10, respectively, which blocked Notch activation, prevented IL-10 secretion. HCs from Con A–pretreated mice showed enhanced expression of the Notch ligand Jagged1 and significantly increased receptor density of Notch1 on CD4+ T cells. However, HCs from Con A–pretreated IFN regulatory factor 1−/− mice, which cannot respond to IFN-γ, as well as those from IFN-γ−/− mice failed to augment IL-10 production by CD4+ T cells. In conclusion, it seems that HCs fine-tune liver inflammation by upregulation of Jagged1 and activation of Notch signaling in Th1 cells. This mechanism might be of particular importance in the regenerating liver subsequent to Th1-mediated hepatitis.

Polo-like Kinase 2, a Novel ADAM17 Signaling Component, Regulates Tumor Necrosis Factor α Ectodomain Shedding

Background: The metalloprotease ADAM17 emerged as the main sheddase of several cytokines and cytokine receptors. Results: The acidophilic kinase PLK2 interacts with and phosphorylates ADAM17 in mammalian cells. Conclusion: PLK2 represents a novel cellular interaction partner of ADAM17 modulating its activity. Significance: Regulation of ADAM17 activity is essential for inflammatory responses. 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.

Short-term TNFα shedding is independent of cytoplasmic phosphorylation or furin cleavage of ADAM17.

Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNFα (pro-TNFα) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNFα cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNFα shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNFα was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNFα cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNFα as well as of both TNF-receptors Finally, we showed that a pro-TNFα mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNFα cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17.

IL‐6 trans‐signaling is essential for the development of hepatocellular carcinoma in mice

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).