Non-canonical STAT3 function reduces REDD1 transcription.

The transcription factor STAT3 is a potent activator of transcription, but evidence exists that STAT3 can also repress gene expression. However, little is known about the molecular mechanisms involved in STAT3-dependent gene repression. Notably, STAT3 reduces the expression of the stress-induced mTOR inhibitor REDD1 by reducing REDD1 mRNA transcription. Here, we determined the functional domains of STAT3 responsible for the reduction of REDD1 mRNA and protein expression. Within STAT3, the N-terminal domain and tyrosine 705 are crucial for STAT3-dependent reduction of REDD1 expression. Interestingly, binding of STAT3 to canonical STAT-binding sides within the REDD1 promoter is not necessary for STAT3-mediated reduction of REDD1 expression. Still, STAT3 is recruited to the REDD1 promoter upon stimulation with IL-6, and reduces REDD1 promoter activity. The reduction of REDD1 expression is specific for STAT3, as neither expression nor activation of STAT1 reduces REDD1 mRNA and protein expression. In summary, we present a novel, non-canonical STAT3-dependent mechanism for reducing gene expression. This transcriptional repression increases the functions of STAT3 proteins beyond classical transcriptional activation of cytokine-regulated target genes to a more complex function in modulating gene expression in immunity and cellular stress.

Robustness and Information Transfer within IL-6-induced JAK/STAT Signalling.

Cellular communication via intracellular signalling pathways is crucial. Expression and activation of signalling proteins is heterogenous between isogenic cells of the same cell-type. However, mechanisms evolved to enable sufficient communication and to ensure cellular functions. We use information theory to clarify mechanisms facilitating IL-6-induced JAK/STAT signalling despite cell-to-cell variability. We show that different mechanisms enabling robustness against variability complement each other. Early STAT3 activation is robust as long as cytokine concentrations are low. Robustness at high cytokine concentrations is ensured by high STAT3 expression or serine phosphorylation. Later the feedback-inhibitor SOCS3 increases robustness. Channel Capacity of JAK/STAT signalling is limited by cell-to-cell variability in STAT3 expression and is affected by the same mechanisms governing robustness. Increasing STAT3 amount increases Channel Capacity and robustness, whereas increasing STAT3 tyrosine phosphorylation reduces robustness but increases Channel Capacity. In summary, we elucidate mechanisms preventing dysregulated signalling by enabling reliable JAK/STAT signalling despite cell-to-cell heterogeneity.

The Fusarium toxin deoxynivalenol disrupts phenotype and function of monocyte-derived dendritic cells in vivo and in vitro.

The trichothecene mycotoxin deoxynivalenol (DON) causes systemic immuno-suppression in pigs and possibly also in humans after chronic dietary exposure. Since the outcome of every immune response is largely controlled by dendritic cells (DC), we hypothesised that a direct influence of DON on DC function might play a role in mediating DON immunotoxicity. To test this hypothesis, a 2x2 factorial design study was performed. Pigs were fed a control diet or a diet containing DON (DON-diet); monocyte-derived DC (MoDC) from these pigs were then treated with DON in vitro or left untreated. Phenotype and function of the MoDC were analysed. In vitro DON-treatment of MoDC from pigs fed the control diet resulted in a down-regulation of CD80/86 and CD40. This was associated with an activation of the mitogen-associated protein kinases ERK1/2 and JNK. The endocytic activity of MoDC was decreased after in vitro DON-exposure while their T cell stimulatory capacity was not altered. MoDC derived from pigs that had been fed the DON-diet failed to up-regulate MHC-II in response to LPS/TNFalpha. Dietary exposure of pigs to DON inhibited endocytosis of FITC-dextran by MoDC, but did not influence T cell stimulatory capacity. ERK1/2 and JNK were constitutively activated in MoDC from pigs fed the DON-diet. If MoDC derived from pigs fed the DON-diet were exposed to DON in vitro, this resulted in an up-regulation of MHC-II and CD80/86, but not CD40. In comparison to untreated MoDC from pigs fed DON-diet, endocytic capacity was further down-regulated, whereas mitogen-activated protein kinase activation was increased. In summary, DON disrupts porcine DC function in vitro and in vivo, which might contribute to the immunosuppressive effects of this mycotoxin.

Interleukin-6 influences stress-signalling by reducing the expression of the mTOR-inhibitor REDD1 in a STAT3-dependent manner.

Interleukin 6 (IL-6) is a pleiotropic cytokine and a strong activator of Mammalian Target of Rapamycin (mTOR). In contrast, mTOR activity is negatively regulated by Regulated in Development and DNA Damage Responses 1 (REDD1). Expression of REDD1 is induced by cellular stressors such as glucocorticoids and DNA damaging agents. We show that the expression of basal as well as stress-induced REDD1 is reduced by IL-6. The reduction of REDD1 expression by IL-6 is independent of proteasomal or caspase-mediated degradation of REDD1 protein. Instead, induction of REDD1 mRNA is reduced by IL-6. The regulation of REDD1 expression by IL-6 is independent of Phosphatidylinositide-3-Kinase (PI3K) and Mitogen-Activated Protein Kinase (MAPK) signalling but depends on the expression and activation of Signal Transducer and Activator of Transcription 3 (STAT3). Furthermore, the reduction of basal REDD1 expression by IL-6 correlates with IL-6-induced activation of mTOR signalling. Inhibition of STAT3 activation blocks IL-6-induced mTOR activation. In summary, we present a novel STAT3-dependent mechanism of both IL-6-induced activation of mTOR and IL-6-dependent reversion of stress-induced inhibition of mTOR activity.

Host-pathogen systems biology: logical modelling of hepatocyte growth factor and Helicobacter pylori induced c-Met signal transduction.

BACKGROUND: The hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of tissues, including epithelial cells, on binding to the receptor tyrosine kinase c-Met. Abnormal c-Met signalling contributes to tumour genesis, in particular to the development of invasive and metastatic phenotypes. The human microbial pathogen Helicobacter pylori can induce chronic gastritis, peptic ulceration and more rarely, gastric adenocarcinoma. The H. pylori effector protein cytotoxin associated gene A (CagA), which is translocated via a type IV secretion system (T4SS) into epithelial cells, intracellularly modulates the c-Met receptor and promotes cellular processes leading to cell scattering, which could contribute to the invasiveness of tumour cells. Using a logical modelling framework, the presented work aims at analysing the c-Met signal transduction network and how it is interfered by H. pylori infection, which might be of importance for tumour development. RESULTS: A logical model of HGF and H. pylori induced c-Met signal transduction is presented in this work. The formalism of logical interaction hypergraphs (LIH) was used to construct the network model. The molecular interactions included in the model were all assembled manually based on a careful meta-analysis of published experimental results. Our model reveals the differences and commonalities of the response of the network upon HGF and H. pylori induced c-Met signalling. As another important result, using the formalism of minimal intervention sets, phospholipase Cgamma1 (PLCgamma1) was identified as knockout target for repressing the activation of the extracellular signal regulated kinase 1/2 (ERK1/2), a signalling molecule directly linked to cell scattering in H. pylori infected cells. The model predicted only an effect on ERK1/2 for the H. pylori stimulus, but not for HGF treatment. This result could be confirmed experimentally in MDCK cells using a specific pharmacological inhibitor against PLCgamma1. The in silico predictions for the knockout of two other network components were also verified experimentally. CONCLUSION: This work represents one of the first approaches in the direction of host-pathogen systems biology aiming at deciphering signalling changes brought about by pathogenic bacteria. The suitability of our network model is demonstrated by an in silico prediction of a relevant target against pathogen infection.

Cholera toxin promotes the generation of semi-mature porcine monocyte-derived dendritic cells that are unable to stimulate T cells.

Cholera toxin (Ctx) is a powerful mucosal adjuvant with potential applications for oral vaccination of swine. Dendritic cells (DC) play a key role in the decision between immunity and tolerance, and are likely target cells for mediating Ctx functions in vivo. Therefore, we examined the capacity of Ctx to enhance stimulatory activity of porcine monocyte-derived DC (MoDC). Ctx promoted the development of a semi-mature DC phenotype, with decreased levels of MHC class II and CD40, but increased CD80/86 expression. These changes were associated with activation of extracellular signal-regulated kinase (ERK), but not NFkappaB or c-Jun N-terminal kinase (JNK). Functionally, Ctx-priming greatly diminished T cell stimulatory capacity both in antigen-specific and superantigen-induced proliferation assays. The lower proliferation rate was not due to increased apoptosis of either DC or T cells. Ctx suppressed TNFalpha secretion by MoDC, but induced IL-10 production. The observed effects on T cell proliferation could only be partially mimicked by IL-10 alone. However, addition of recombinant TNFalpha to co-cultures of Ctx-primed MoDC and lymphocytes restored lymphocyte proliferation in a concentration-dependent manner. Ctx-primed DC were not actively tolerogenic, since they could not suppress proliferative T cell reactions induced by untreated DC.

Receptor endocytosis counteracts the development of opioid tolerance.

In contrast to endogenous opioids, the highly addictive drug morphine activates the mu-opioid receptor without causing its rapid endocytosis. It has recently been reported that coapplication of low concentrations of [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) facilitates the ability of morphine to stimulate mu-opioid receptor endocytosis and prevents the development of morphine tolerance in rats. To investigate the clinical relevance of this finding for analgesic therapy, the endocytotic efficacies of a series of clinically used opioids were determined, and the effect of a combination of these drugs with morphine on the mu-opioid receptor endocytosis in receptor-expressing human embryonic kidney (HEK) 293 cells was quantified. The combination of morphine and opioid drugs with high endocytotic efficacies (e.g., DAMGO, etonitazene, sufentanil, beta-endorphin, piritramide, or methadone) did not result in a facilitation of morphine-mediated endocytosis but rather in a decrease of the receptor endocytosis mediated by the tested opioid drugs. These findings demonstrate a partial agonistic effect of morphine on the agonist-induced receptor endocytosis. Moreover, we demonstrated that the endocytotic potencies of opioid drugs are negatively correlated with their ability to cause receptor desensitization and opioid tolerance in HEK 293 cells. These results strongly support the hypothesis that mu-opioid receptor endocytosis counteracts receptor desensitization and opioid tolerance by inducing fast receptor reactivation and recycling. In addition, it is shown that agonist-induced receptor endocytosis facilitates the compensatory up-regulation of the cAMP pathway, a cellular hallmark of opioid withdrawal. Our findings suggest that opioids with high endocytotic efficacies might cause reduced opioid tolerance but can facilitate compensatory mechanisms, resulting in an enhanced opioid dependence.