Crosstalk between different family members: IL27 recapitulates IFNγ responses in HCC cells, but is inhibited by IL6-type cytokines.

Interleukin-27 (IL27) is a type-I-cytokine of the IL6/IL12 family predominantly secreted by activated macrophages and dendritic cells. In the liver, IL27 expression was observed to be upregulated in patients with hepatitis B, and sera of hepatocellular carcinoma (HCC) patients contain significantly elevated levels of IL27 compared to healthy controls or patients with hepatitis and/or liver cirrhosis. In this study, we show that IL27 induces STAT1 and STAT3 phosphorylation in 5 HCC lines and 3 different types of non-transformed liver cells. We were especially interested in the relevance of the IL27-induced STAT3 activation in liver cells. Thus, we compared the IL27 responses with those induced by IFNγ (STAT1-dominated response) or IL6-type cytokines (IL6, hyper-IL6 (hy-IL6) or OSM) (STAT3-dominated response) by microarray analysis and find that in HCC cells, IL27 induces an IFNγ-like, STAT1-dependent transcriptional response, but we do not find an effective STAT3-dependent response. Validation experiments corroborate the finding from the microarray evaluation. Interestingly, the availability of STAT1 seems critical in the shaping of the IL27 response, as the siRNA knock-down of STAT1 revealed the ability of IL27 to induce the acute-phase protein γ-fibrinogen, a typical IL6 family characteristic. Moreover, we describe a crosstalk between the signaling of IL6-type cytokines and IL27: responses to the gp130-engaging cytokine IL27 (but not those to IFNs) can be inhibited by IL6-type cytokine pre-stimulation, likely by a SOCS3-mediated mechanism. Thus, IL27 recapitulates IFNγ responses in liver cells, but differs from IFNγ by its sensitivity to SOCS3 inhibition.

A new ALK isoform transported by extracellular vesicles confers drug resistance to melanoma cells.

BACKGROUND: Drug resistance remains an unsolved clinical issue in oncology. Despite promising initial responses obtained with BRAF and MEK kinase inhibitors, resistance to treatment develops within months in virtually all melanoma patients. METHODS: Microarray analyses were performed in BRAF inhibitor-sensitive and resistant cell lines to identify changes in the transcriptome that might play a role in resistance. siRNA approaches and kinase inhibitors were used to assess the involvement of the identified Anaplastic Lymphoma Kinase (ALK) in drug resistance. The capability of extracellular vesicles (EVs) to transfer drug resistant properties was investigated in co-culture assays. RESULTS: Here, we report a new mechanism of acquired drug resistance involving the activation of a novel truncated form of ALK. Knock down or inhibition of ALK re-sensitised resistant cells to BRAF inhibition and induced apoptosis. Interestingly, truncated ALK was also secreted into EVs and we show that EVs were the vehicle for transferring drug resistance. CONCLUSIONS: To our knowledge, this is the first report demonstrating the functional involvement of EVs in melanoma drug resistance by transporting a truncated but functional form of ALK, able to activate the MAPK signalling pathway in target cells. Combined inhibition of ALK and BRAF dramatically reduced tumour growth in vivo. These findings make ALK a promising clinical target in melanoma patients.

Oncostatin M induces RIG-I and MDA5 expression and enhances the double-stranded RNA response in fibroblasts.

Interleukin (IL)-6-type cytokines have no direct antiviral activity; nevertheless, they display immune-modulatory functions. Oncostatin M (OSM), a member of the IL-6 family, has recently been shown to induce a distinct number of classical interferon stimulated genes (ISG). Most of them are involved in antigen processing and presentation. However, induction of retinoic acid-inducible gene (RIG)-I-like receptors (RLR) has not been investigated. Here we report that OSM has the capability to induce the expression of the DExD/H-Box RNA helicases RIG-I and melanoma differentiation antigen 5 (MDA5) as well as of the transcription factors interferon regulatory factor (IRF)1, IRF7 and IRF9 in primary fibroblasts. Induction of the helicases depends on tyrosine as well as serine phosphorylation of STAT1. Moreover, we could show that the OSM-induced STAT1 phosphorylation is predominantly counter-regulated by a strong STAT3-dependent SOCS3 induction, as Stat3 as well as Socs3 knock-down results in an enhanced and prolonged helicase and IRF expression. Other factors involved in regulation of STAT1 or IRF1 activity, like protein tyrosine phosphatase, non-receptor type 2 (PTPN2), promyelocytic leukaemia protein (PML) or small ubiquitin-related modifier 1 (SUMO1), play a minor role in OSM-mediated induction of RLR. Remarkably, OSM and interferon-γ (IFN-γ) synergize to mediate transcription of RLR and pre-treatment of fibroblasts with OSM fosters the type I interferon production in response to a subsequent encounter with double-stranded RNA. Together, these findings suggest that the OSM-induced JAK/STAT1 signalling is implicated in virus protection of non-professional immune cells and may cooperate with interferons to enhance RLR expression in these cells.

ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells.

BACKGROUND: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism. METHODS: Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. RESULTS: We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation. Suppression of ROS by MitoQ suppressed PDH-E1α phosphorylation, strongly suggesting that ROS mediate the activation of PDKs. Interestingly, the inhibition of PDK1 with AZD7545 specifically suppressed growth of BRAF-mutant and BRAF inhibitor resistant melanoma cells. CONCLUSIONS: In BRAFV600E and BRAFWT/NRASmut melanoma cells, the increased production of ROS upon inhibition of the RAS/RAF/MEK/ERK pathway, is responsible for activating PDKs, which in turn phosphorylate and inactivate PDH. As part of a possible salvage pathway, the tricarboxylic acid cycle is inhibited leading to reduced oxidative metabolism and reduced ROS levels. We show that inhibition of PDKs by AZD7545 leads to growth suppression of BRAF-mutated and -inhibitor resistant melanoma cells. Thus small molecule PDK inhibitors such as AZD7545, might be promising drugs for combination treatment in melanoma patients with activating RAS/RAF/MEK/ERK pathway mutations (50% BRAF, 25% NRASmut, 11.9% NF1mut).

Cooperative effects of Janus and Aurora kinase inhibition by CEP701 in cells expressing Jak2V617F.

The Janus kinase 2 mutant V617F occurs with high frequency in myeloproliferative neoplasms. Further mutations affecting the Janus kinase family have been discovered mostly in leukaemias and in myeloproliferative neoplasms. Owing to their involvement in neoplasia, inflammatory diseases and in the immune response, Janus kinases are promising targets for kinase inhibitor therapy in these disease settings. Various quantitative assays including two newly developed screening assays were used to characterize the function of different small-molecule compounds in cells expressing Jak2V617F. A detailed comparative analysis of different Janus kinase inhibitors in our quantitative assays and the subsequent characterization of additional activities demonstrated for the first time that the most potent Jak2 inhibitor in our study, CEP701, also targets Aurora kinases. CEP701 shows a unique combination of both activities which is not found in other compounds also targeting Jak2. Furthermore, colony forming cell assays showed that Janus kinase 2 inhibitors preferentially suppressed the growth of erythroid colonies, whereas inhibitors of Aurora kinases preferentially blocked myeloid colony growth. CEP701 demonstrated a combined suppression of both colony types. Moreover, we show that combined application of a Janus and an Aurora kinase inhibitor recapitulated the effect observed for CEP701 but might allow for more flexibility in combining both activities in clinical settings, e.g. in the treatment of myeloproliferative neoplasms. The newly developed screening assays are high throughput compatible and allow an easy detection of new compounds with Janus kinase 2 inhibitory activity.

CCT196969 effectively inhibits growth and survival of melanoma brain metastasis cells.

Melanomas frequently metastasize to the brain. Despite recent progress in the treatment of melanoma brain metastasis, therapy resistance and relapse of disease remain unsolved challenges. CCT196969 is a SRC family kinase (SFK) and Raf proto-oncogene, serine/threonine kinase (RAF) inhibitor with documented effects in primary melanoma cell lines in vitro and in vivo. Using in vitro cell line assays, we studied the effects of CCT196969 in multiple melanoma brain metastasis cell lines. The drug effectively inhibited proliferation, migration, and survival in all examined cell lines, with viability IC50 doses in the range of 0.18-2.6 µM. Western blot analysis showed decreased expression of p-ERK, p-MEK, p-STAT3 and STAT3 upon CCT196969 treatment. Furthermore, CCT196969 inhibited viability in two B-Raf Proto-Oncogene (BRAF) inhibitor resistant metastatic melanoma cell lines. Further in vivo studies should be performed to determine the treatment potential of CCT196969 in patients with treatment-naïve and resistant melanoma brain metastasis.

An unusual insertion in Jak2 is crucial for kinase activity and differentially affects cytokine responses.

The Janus kinases, Jaks, constitutively associate with the cytoplasmic region of cytokine receptors and play an important role in a multitude of biological processes. Jak2 dysfunction has been implicated in myeloproliferative diseases and leukemia. Although Jaks were studied extensively for many years, the molecular mechanism of Jak activation upon cytokine stimulation of cells is still incompletely understood. In this study, we investigated the importance of an unusual insertion located within the kinase domain in Jak2. We found that the deletion of this insertion, which we named the Jak-specific insertion (JSI), totally abrogates Jak2 autophosphorylation. We further point mutated four residues within the JSI that are conserved in all Jak family members. Three of these mutants showed abrogated or reduced autophosphorylation, whereas the fourth displayed increased autophosphorylation. We found that the phosphorylation state of these mutants is not influenced by other domains of the kinase. Our data further suggest that the JSI is not required for the negative regulation of kinase activity by the suppressor of cytokine signaling proteins, SOCS. Most importantly, we show that mutations in this region differentially affect IFN-gamma and erythropoietin signal transduction. Taken together, the dramatic effects on the phosphorylation status of Jak2 as well as the differential effects on the signaling via different cytokines highlight the importance of this unusual region for the catalytic activity of Jaks.

Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases.

Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets.

Hypoxia-inducible factor 1alpha is up-regulated by oncostatin M and participates in oncostatin M signaling.

UNLABELLED: The interleukin-6-type cytokine oncostatin M (OSM) acts via the Janus kinase/signal transducer and activator of transcription pathway as well as via activation of mitogen-activated protein kinases and is known to critically regulate processes such as liver development and regeneration, hematopoiesis, and angiogenesis, which are also determined by hypoxia with the hypoxia-inducible factor 1alpha (HIF1alpha) as a key component. Here we show that treatment of hepatocytes and hepatoma cells with OSM leads to an increased protein level of HIF1alpha under normoxic and hypoxic conditions. Furthermore, the OSM-dependent HIF1alpha increase is mediated via Janus kinase/signal transducer and activator of transcription 3 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 pathways. OSM-mediated HIF1alpha up-regulation did not result from an increase in HIF1alpha protein stability but from increased transcription from the HIF1alpha gene. In addition, we show that the OSM-induced HIF1alpha gene transcription and the resulting enhanced HIF1alpha protein levels are important for the OSM-dependent vascular endothelial growth factor and plasminogen activator inhibitor 1 gene induction associated with several diseases. CONCLUSION: HIF1alpha levels increase significantly after treatment of hepatocytes and hepatoma cells with OSM, and HIF1alpha contributes to OSM downstream signaling events, pointing to a cross-talk between cytokine and hypoxia signaling in processes such as liver development and regeneration.

Interleukin-27 displays interferon-gamma-like functions in human hepatoma cells and hepatocytes.

UNLABELLED: Interleukin-27 (IL-27) is a cytokine belonging to the IL-6/IL-12 cytokine family. It is secreted by antigen-presenting cells, strongly acts on T cells, and also stimulates innate immune cells. In most studies, the effects of IL-27 on T cells were investigated; however, not much is known about possible effects of IL-27 on other cell types. IL-27 signals via the common IL-6-type cytokine receptor chain gp130 and the IL-27-specific chain WSX-1. Given the importance of gp130 in regulating liver responses such as the acute phase response or liver regeneration, we investigated whether IL-27 could also have a function in liver cells. We find that IL-27 stimulates hepatoma cells and hepatocytes by inducing a sustained signal transducer and activator of transcription (STAT)1 and STAT3 activation. Whereas the STAT3 mediated responses to IL-27 (gamma-fibrinogen and hepcidin induction) are not detectable, we observe an interferon-gamma (IFN-gamma)-like STAT1 response leading to the induction of interferon-regulated proteins such as STAT1, STAT2, interferon response factor (IRF)-1, IRF-9, myxovirus resistance A and guanylate binding protein 2. CONCLUSION: Our study provides evidence for a function of IL-27 in hepatoma cells and hepatocytes and shows that IL-27 responses are not restricted to the classical immune cells. Our results suggest that IL-27 exerts IFN-like functions in liver cells and that it can contribute to the antiviral response in these cells.

Oncostatin M-induced and constitutive activation of the JAK2/STAT5/CIS pathway suppresses CCL1, but not CCL7 and CCL8, chemokine expression.

The recruitment of leukocytes to injured tissue is crucial for the initiation of inflammatory responses as well as for immune surveillance to fight tumor progression. In this study, we show that oncostatin M, a member of the IL-6-type cytokine family and potent proinflammatory cytokine stimulates the expression of the chemokines CCL1, CCL7, and CCL8 in primary human dermal fibroblasts at a faster kinetic than IL-1beta or TNF-alpha. The production of CCL1 and CCL8 is important for migration of monocytes, while specific Abs against CCL1 additionally inhibit the migration of T lymphocytes. We identify the mitogen-activated protein kinases ERK1/2 and p38 as crucial factors for the enhanced expression of CCL1 and CCL8. Depletion of the ERK1/2 target genes c-Jun or c-Fos strongly decrease CCL1 and CCL8 expression, while p38 MAPK prolongs the half-life of CCL1, CCL7, and CCL8 mRNA through inhibition of tristetraprolin. None of the STAT transcription factors STAT1, STAT3, or STAT5 stimulate transcription of CCL1 or CCL8. However, we identify a negative regulatory function of activated STAT5 for the gene expression of CCL1. Importantly, not STAT5 itself, but its target gene cytokine inducible SH2-domain containing protein is required for the STAT5 inhibitory effect on CCL1 expression. Finally, we show that constitutive activation of STAT5 through a mutated form of JAK2 (JAK2 V617F) occurring in patients with myeloproliferative disorders similarly suppresses CCL1 expression. Taken together, we identify novel important inflammatory target genes of OSM which are independent of STAT signaling per se, but depend on MAPK activation and are partly repressed through STAT5-dependent expression of cytokine inducible SH2-domain containing protein.

Systematic Transcriptional Profiling of Responses to STAT1- and STAT3-Activating Cytokines in Different Cancer Types.

Cytokines orchestrate responses to pathogens and in inflammatory processes, but they also play an important role in cancer by shaping the expression levels of cytokine response genes. Here, we conducted a large profiling study comparing miRNome and mRNA transcriptome data generated following different cytokine stimulations. Transcriptomic responses to STAT1- (IFNγ, IL-27) and STAT3-activating cytokines (IL6, OSM) were systematically compared in nine cancerous and non-neoplastic cell lines of different tissue origins (skin, liver and colon). The largest variation in our datasets was seen between cell lines of the three different tissues rather than stimuli. Notably, the variability in miRNome datasets was a lot more pronounced than in mRNA data. Our data also revealed that cells of skin, liver and colon tissues respond very differently to cytokines and that the cell signaling networks activated or silenced in response to STAT1- or STAT3-activating cytokines are specific to the tissue and the type of cytokine. However, globally, STAT1-activating cytokines had stronger effects than STAT3-inducing cytokines with most significant responses in liver cells, showing more genes upregulated and with higher fold change. A more detailed analysis of gene regulations upon cytokine stimulation in these cells provided insights into STAT1- versus STAT3-driven processes in hepatocarcinogenesis. Finally, independent component analysis revealed interconnected transcriptional networks distinct between cancer cells and their healthy counterparts.

Many ways to resistance: How melanoma cells evade targeted therapies.

Melanoma is an aggressive malignancy originating from pigment-producing melanocytes. The development of targeted therapies (MAPK pathway inhibitors) and immunotherapies (immune checkpoint inhibitors) led to a substantial improvement in overall survival of patients. However, the long-term efficacy of such treatments is limited by side effects, lack of clinical effects and the rapidly emerging resistance to treatment. A number of molecular mechanisms underlying this resistant phenotype have already been elucidated. In this review, we summarise currently available treatment options for metastatic melanoma and the known resistance mechanisms to targeted therapies. A focus will be placed on "phenotype switching" as a mechanism and driver of drug resistance, together with an overview of novel approaches to circumvent resistance. A large body of recent data and literature suggests that tumour progression and phenotype switching could be better controlled and development of resistance prevented or at least delayed, by combining drugs targeting fast- and slow-proliferating cells.

Modulation of the IL-6-Signaling Pathway in Liver Cells by miRNAs Targeting gp130, JAK1, and/or STAT3.

Interleukin-6 (IL-6)-type cytokines share the common receptor glycoprotein 130 (gp130), which activates a signaling cascade involving Janus kinases (JAKs) and signal transducer and activator of transcription (STAT) transcription factors. IL-6 and/or its signaling pathway is often deregulated in diseases, such as chronic liver diseases and cancer. Thus, the identification of compounds inhibiting this pathway is of interest for future targeted therapies. We established novel cellular screening systems based on a STAT-responsive reporter gene (Cypridina luciferase). Of a library containing 538 microRNA (miRNA) mimics, several miRNAs affected hyper-IL-6-induced luciferase activities. When focusing on candidate miRNAs specifically targeting 3' UTRs of signaling molecules of this pathway, we identified, e.g., miR-3677-5p as a novel miRNA affecting protein expression of both STAT3 and JAK1, whereas miR-16-1-3p, miR-4473, and miR-520f-3p reduced gp130 surface expression. Interestingly, combination treatment with 2 or 3 miRNAs targeting gp130 or different signaling molecules of the pathway did not increase the inhibitory effects on phospho-STAT3 levels and STAT3 target gene expression compared to treatment with single mimics. Taken together, we identified a set of miRNAs of potential therapeutic value for cancer and inflammatory diseases, which directly target the expression of molecules within the IL-6-signaling pathway and can dampen inflammatory signal transduction.

Kinase inhibitor library screening identifies synergistic drug combinations effective in sensitive and resistant melanoma cells.

BACKGROUND: Melanoma is the most aggressive and deadly form of skin cancer with increasing case numbers worldwide. The development of inhibitors targeting mutated BRAF (found in around 60% of melanoma patients) has markedly improved overall survival of patients with late-stage tumors, even more so when combined with MEK inhibitors targeting the same signaling pathway. However, invariably patients become resistant to this targeted therapy resulting in rapid progression with treatment-refractory disease. The purpose of this study was the identification of new kinase inhibitors that do not lead to the development of resistance in combination with BRAF inhibitors (BRAFi), or that could be of clinical benefit as a 2nd line treatment for late-stage melanoma patients that have already developed resistance. METHODS: We have screened a 274-compound kinase inhibitor library in 3 BRAF mutant melanoma cell lines (each one sensitive or made resistant to 2 distinct BRAFi). The screening results were validated by dose-response studies and confirmed the killing efficacies of many kinase inhibitors. Two different tools were applied to investigate and quantify potential synergistic effects of drug combinations: the Chou-Talalay method and the Synergyfinder application. In order to exclude that resistance to the new treatments might occur at later time points, synergistic combinations were administered to fluorescently labelled parental and resistant cells over a period of > 10 weeks. RESULTS: Eight inhibitors targeting Wee1, Checkpoint kinase 1/2, Aurora kinase, MEK, Polo-like kinase, PI3K and Focal adhesion kinase killed melanoma cells synergistically when combined with a BRAFi. Additionally, combination of a Wee1 and Chk inhibitor showed synergistic killing effects not only on sensitive cell lines, but also on intrinsically BRAFi- and treatment induced-resistant melanoma cells. First in vivo studies confirmed these observations. Interestingly, continuous treatment with several of these drugs, alone or in combination, did not lead to emergence of resistance. CONCLUSIONS: Here, we have identified new, previously unexplored (in the framework of BRAFi resistance) inhibitors that have an effect not only on sensitive but also on BRAFi-resistant cells. These promising combinations together with the new immunotherapies could be an important step towards improved 1st and 2nd line treatments for late-stage melanoma patients.

The PD-L1- and IL6-mediated dampening of the IL27/STAT1 anticancer responses are prevented by α-PD-L1 or α-IL6 antibodies.

Interleukin-27 (IL27) is a type-I cytokine of the IL6/IL12 family and is predominantly secreted by activated macrophages and dendritic cells. We show that IL27 induces STAT factor phosphorylation in cancerous cell lines of different tissue origin. IL27 leads to STAT1 phosphorylation and recapitulates an IFN-γ-like response in the microarray analyses, with up-regulation of genes involved in antiviral defense, antigen presentation, and immune suppression. Like IFN-γ, IL27 leads to an up-regulation of TAP2 and MHC-I proteins, which mediate increased tumor immune clearance. However, both cytokines also upregulate proteins such as PD-L1 (CD274) and IDO-1, which are associated with immune escape of cancer. Interestingly, differential expression of these genes was observed within the different cell lines and when comparing IL27 to IFN-γ. In coculture experiments of hepatocellular carcinoma (HCC) cells with peripheral blood mononuclear cells, pre-treatment of the HCC cells with IL27 resulted in lowered IL2 production by anti-CD3/-CD28 activated T-lymphocytes. Addition of anti-PD-L1 antibody, however, restored IL2 secretion. The levels of other TH 1 cytokines were also enhanced or restored upon administration of anti-PD-L1. In addition, we show that the suppression of IL27 signaling by IL6-type cytokine pre-stimulation-mimicking a situation occurring, for example, in IL6-secreting tumors or in tumor inflammation-induced cachexia-can be antagonized by antibodies against IL6-type cytokines or their receptors. Therapeutically, the antitumor effects of IL27 (mediated, e.g., by increased antigen presentation) might thus be increased by combining IL27 with blocking antibodies against PD-L1 or/and IL6-type cytokines.

A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background.

We compared several alternative ECL solutions for Western blot detection of endogenous proteins in whole cell lysates using inexpensive, commercially available reagents. Starting from an existing protocol based on p-coumaric acid (pCA) as enhancer, we found that the ECL solution containing 4-iodophenylboronic acid (4IPBA) generated strong specific signals and low background chemiluminescence. We optimised the luminol, 4IPBA and hydrogenperoxide concentrations of this 4IPBA-ECL solution. The optimised 4IPBA-ECL solution (100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 2 mM 4IPBA, 5.3 mM hydrogenperoxide) shows a greatly increased signal intensity compared to the initial pCA-ECL protocol and to some commercially available ECL solutions. In addition, the optimised 4IPBA-ECL solution also generates much lower background chemiluminescence than other non-commercial ECL solutions using p-coumaric acid or 4-iodophenol as enhancers. The 4IPBA-ECL solution was stable when stored but had the lowest background when prepared freshly from stock solutions. Thus, we present an optimised protocol for a well-performing inexpensive ECL solution which is an alternative to expensive commercial ECL solutions and which achieves a better signal and lower background than the commercial solutions tested.

The multi-site docking protein Gab1 is constitutively phosphorylated independent from its recruitment to the plasma membrane in Jak2-V617F-positive cells and mediates proliferation of human erythroleukaemia cells.

The constitutively active Janus kinase 2 mutant Jak2-V617F is responsible for cytokine-independent growth of hematopoietic cells and the development of myeloproliferative neoplasms, such as polycythaemia vera and essential thrombocythaemia. Cells expressing Jak2-V617F exhibit constitutive STAT, MAPK, and PI3K signalling, and constitutive association of the multi-site docking protein Gab1 to PIP3 at the plasma membrane. Here, we demonstrate the crucial role of Gab1 for the proliferation of Jak2-V617F-positive human erythroleukaemia (HEL) cells. In Jak2-V617F-expressing cells Gab1 is constitutively phosphorylated by Erk1/2 on serine residue 552, which regulates binding to PIP3. Additionally, Gab1 is constitutively phosphorylated on tyrosine residue 627. Tyrosine 627 is a SHP2 binding site and required for Gab1-dependent Erk1/2 activation. As previously shown, Jak2-V617F-dependent Erk1/2 and PI3K activation act synergistically on the proliferation of Jak2-V617F-positive cells. Here, we examined whether constitutive membrane association of Gab1 explains cytokine-independent Gab1 phosphorylation in Jak2-V617F-expressing cells. Although we could demonstrate Jak2-V617F-dependent constitutive serine 552 and tyrosine 627 phosphorylation of Gab1, interestingly, both phosphorylations do not require binding of Gab1 to PIP3 at the plasma membrane. Instead, we observed a constitutive interaction of Gab1 with the erythropoietin receptor in Jak2-V617F-expressing cells, which depends on Janus kinase activity. Thus, constitutive Gab1-dependent signalling in Jak2-V617F-expressing cells does not occur due to the constitutive association of Gab1 with PIP3 at the plasma membrane.

Jaks and cytokine receptors--an intimate relationship.

Most cytokine receptors lack intrinsic kinase activity and many of them signal via Janus kinases (Jaks). These tyrosine kinases are associated with cytokine receptor subunits, they become activated upon receptor triggering and subsequently activate downstream signalling events, e.g. the phosphorylation of STAT transcription factors. The successful interplay between cytokines, their receptors and the connected Jaks not only determines signalling competence but is also vital for intracellular traffic, stability, and fate of the cognate receptors. Here, we will discuss underlying mechanisms as well as some structural features with a focus on Jak1 and two of the signal transducing receptor subunits of interleukin (IL)-6 type cytokines, gp130 and OSMR. Regions that are critically involved in Jak-binding have been identified for many cytokine receptor subunits. In most cases the membrane-proximal parts comprising the box1 and box2 regions within the receptor are involved in this association while, within Jaks, the N-terminal FERM domain, possibly together with the SH2-like domain, are pivotal for binding to the relevant receptors. The exclusive membrane localisation of Jaks depends on their ability to associate with cytokine receptors. For gp130 and Jak1, it was shown that the cytokine receptor/Jak complex can be regarded as a receptor tyrosine kinase since both molecules have the same diffusion dynamics and are virtually undissociable. Furthermore, Jaks take an active role in the regulation of the surface expression of at least some cytokine receptors, including the OSMR and this may provide a quality control mechanism ensuring that only signalling-competent receptors (i.e. those with an associated Jak) would be enriched at the cell surface.

Multiple reasons for an inefficient STAT1 response upon IL-6-type cytokine stimulation.

IL-6-type cytokines play an important role during inflammation and the immune response. In addition, they are involved in haematopoiesis, liver and neuronal regeneration, embryonic development and fertility. We found that IL-6-type cytokine stimulation of cell lines and primary human macrophages results in a different distribution of the DNA-binding competent STAT dimer species in the cytosol and nucleus as demonstrated by electrophoretic mobility shift assays. In the absence of detergent, STAT3/STAT3, STAT1/STAT3 were the predominant species in the cytoplasm while STAT3/STAT3 was predominant in the nucleus. However, in detergent containing total cellular lysates and nuclear fractions prepared with detergent containing buffers, the STAT1/STAT1 homodimer was as prominent or even more prominent than STAT3/STAT3 and STAT1/STAT3. We were interested in the cause of this discrepancy since STAT1-regulated genes have not been described to be expressed upon IL-6-type cytokine stimulation. In addition to the more transient STAT1 activation, IL-6-type cytokines such as IL-6 and OSM lead to a much less efficient STAT1 activation compared to the potent STAT1 activators IFNgamma and IFNalpha. Studies with STAT1-deficient cells revealed that STAT1 activation does not seem to be an important competitive process to STAT3 activation arguing again for a very inefficient STAT1 activation upon IL-6-type cytokine stimulation. We also describe that pY-STAT3 is much more efficiently shuttled into the nucleus than pY-STAT1.