Anna Dittrich

Interleukin-6 signalling: More than Jaks and STATs

The hallmark of signalling by many cytokines is the activation of the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathway. However, cytokines additionally activate other pathways. In past years we realised that these pathways significantly contribute to the physiological functions of IL-6 and pathophysiological functions in the context of many inflammatory and proliferative diseases. Whereas other articles in this issue of the European Journal of Cell Biology focus on STAT activation and its regulation we here aim to summarise our knowledge and some remaining questions on interleukin-6 (IL-6)-induced STAT-independent pathways as well as the cross-talk with the Jak/STAT pathway. In the early stages of studying cytokine signalling we were used to analysing individual signalling pathways. These days we know about the importance of both, the crosstalk between pathways initiated by combinations of cytokines as well as the crosstalk between individual pathways initiated by a single cytokine. Whereas the inter-cytokine crosstalk can be studied relatively easily, more sophisticated experimental approaches are required to elucidate the intra-cytokine crosstalk.

Glucocorticoids increase interleukin‐6–dependent gene induction by interfering with the expression of the suppressor of cytokine signaling 3 feedback inhibitor

Glucocorticoids are known to be potent regulators of inflammation and have been used pharmacologically against inflammatory, immune, and lymphoproliferative diseases for more than 50 years. Due to their possible and well‐documented side effects, it is crucial to understand the molecular mechanisms and targets of glucocorticoid action in detail. Several modes of action have been discussed; nevertheless, none of them fully explain all the functions of glucocorticoids. Therefore, we analyzed the cross‐talk between glucocorticoids and interleukin‐6 (IL‐6) in the liver. IL‐6 exerts pro‐inflammatory as well as anti‐inflammatory properties and is a main inducer of the acute‐phase response. The balance between the proinflammatory and anti‐inflammatory activities of IL‐6 is tightly regulated by suppressor of cytokine signaling 3 (SOCS3), a well‐known feedback inhibitor of IL‐6 signaling. Here, it is demonstrated that glucocorticoids enhance IL‐6–dependent γ‐fibrinogen expression. Studying of the underlying mechanism revealed prolonged activation of signal transducer and activator of transcription 3 (STAT3) caused by down‐regulation of SOCS3 protein expression. Consequently, in SOCS3‐deficient cells glucocorticoids do not affect IL‐6–induced signal transduction. Moreover, in hepatocytes lacking the SOCS3 recruiting motif within gp130, IL‐6–dependent γ‐fibrinogen expression is not influenced by glucocorticoid treatment. Conclusion: Glucocorticoids interfere with IL‐6–induced expression of the feedback inhibitor SOCS3, thereby leading to enhanced expression of acute‐phase genes in hepatocytes. This mechanism contributes to the explanation of how glucocorticoids affect inflammation and acute‐phase gene induction. (HEPATOLOGY 2012;55:256–266)

A simple work flow for biologically inspired model reduction--application to early JAK-STAT signaling.

BackgroundModeling of biological pathways is a key issue in systems biology. When constructing a model, it is tempting to incorporate all known interactions of pathway species, which results in models with a large number of unknown parameters. Fortunately, unknown parameters need not necessarily be measured directly, but some parameter values can be estimated indirectly by fitting the model to experimental data. However, parameter fitting, or, more precisely, maximum likelihood parameter estimation, only provides valid results, if the complexity of the model is in balance with the amount and quality of the experimental data. If this is the case the model is said to be identifiable for the given data. If a model turns out to be unidentifiable, two steps can be taken. Either additional experiments need to be conducted, or the model has to be simplified.ResultsWe propose a systematic procedure for model simplification, which consists of the following steps: estimate the parameters of the model, create an identifiability ranking for the estimated parameters, and simplify the model based on the identifiability analysis results. These steps need to be applied iteratively until the resulting model is identifiable, or equivalently, until parameter variances are small. We choose parameter variances as stopping criterion, since they are concise and easy to interpret. For both, the parameter estimation and the calculation of parameter variances, multi-start parameter estimations are run on a parallel cluster. In contrast to related work in systems biology, we do not suggest simplifying a model by fixing some of its parameters, but change the structure of the model.ConclusionsWe apply the proposed approach to a model of early signaling events in the JAK-STAT pathway. The resulting model is not only identifiable with small parameter variances, but also shows the best trade-off between goodness of fit and model complexity.

Model-driven experimental analysis of the function of SHP-2 in IL-6-induced Jak/STAT signaling.

Misregulated interleukin-6 (IL-6)-induced Jak/STAT signaling contributes to many diseases. Under non-pathological conditions Jak/STAT signaling is tightly regulated by a complex network of regulators. One of these regulators is the protein tyrosine phosphatase SH2-containing phosphatase 2 (SHP2). Although SHP2 is known to be a negative regulator of IL-6-induced Jak/STAT signaling, its exact molecular function is not entirely understood. To elucidate the function of SHP2 in IL-6 signaling we followed a systems biology approach, in which modeling, stepwise model refinement, and experimental analysis are closely linked. We come up with an identifiable model of early Jak/STAT signaling that describes the data and proves to be predictive. The model-based analysis implies that (1) the stepwise association of IL-6 with gp80 and gp130 and (2) STAT3 dimerization at the receptor are essential for the dynamics of early pathway activation, and (3) phosphorylation of SHP2 is nonlinear. Furthermore, the modeling results indicate that SHP2 does not act as a feedback inhibitor in an early phase of IL-6-induced Jak/STAT signaling. However, experimental data reveal that SHP2 exhibits a basal repressory function.

Systems biology of IL-6, IL-12 family cytokines.

Interleukin-6-type cytokines play important roles in the communication between cells of multicellular organisms. They are involved in the regulation of complex cellular processes such as proliferation and differentiation and act as key player during inflammation and immune response. A major challenge is to understand how these complex non-linear processes are connected and regulated. Systems biology approaches are used to tackle this challenge in an iterative process of quantitative experimental and mathematical analyses. Here we review quantitative experimental studies and systems biology approaches dealing with the function of Interleukin-6-type cytokines in physiological and pathophysiological conditions. These approaches cover the analyses of signal transduction on a cellular level up to pharmacokinetic and pharmacodynamic studies on a whole organism level.

Glucagon counteracts interleukin-6-dependent gene expression by redundant action of Epac and PKA

Abstract Inflammation is the biological response to injurious stimuli. In the initial phase of the inflammatory process, interleukin-6 (IL-6) is the main inducer of acute phase protein expression in the liver. A prolonged acute phase response is characterised by a disturbed glucose homeostasis and elevated levels of IL-6, insulin, and counterregulatory hormones such as glucagon. Several studies deal with the impact of IL-6 on glucagon-dependent gene expression. In contrast, only very little is known about the influence of G-protein-coupled receptors on IL-6 signalling. Therefore, the aim of this study is to elucidate the regulation of IL-6-induced gene expression by glucagon. We could reveal a novel mechanism of negative regulation of IL-6-induced MAP kinase activation by glucagon in primary murine hepatocytes. IL-6-dependent induction of the ERK-dependent target gene Tfpi2, coding for a Kunitz-type serine protease inhibitor, was strongly down-regulated by glucagon treatment. Studying the underlying mechanism revealed a redundant action of the signalling molecules exchange protein activated by cyclic AMP (Epac) and protein kinase A. The metabolic hormone glucagon interferes in IL-6-induced gene expression. This observation is indicative for a regulatory role of G-protein-coupled receptors in the IL-6-dependent inflammatory response.

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.

Determination of Protein Turnover Rates in the JAK/STAT Pathway Using a Radioactive Pulse-Chase Approach

The turnover rate of different protein species in a signal transduction network strongly affects the impact of the given species on the outcome of a stimulus. Whereas stable, long-lived proteins mainly account for the transmission of a signal, unstable short-lived species often comprise regulatory functions. Here, we describe a method to determine the half-lives of proteins of the JAK/STAT pathway by a pulse-chase approach in cell culture. First, radioactive labeling with (35)S-methionine is carried out to label newly synthesized proteins (pulse). Subsequently, the dynamics of the decay of these proteins is monitored in the absence of labeled amino acids over a defined time period (chase). For this purpose the protein of interest is isolated by immunoprecipitation from total cell lysates, separated on an SDS-polyacrylamide gel, and subsequently visualized by autoradiography.

JAK-STAT Signalling as Example for a Database-Supported Modular Modelling Concept

We present a detailed model of the JAK-STAT pathway in IL-6 signaling as non-trivial case study demonstrating a new database-supported modular modeling method. A module is a self-contained and autonomous Petri net, centred around an individual protein. The modelling approach allows to easily generate and modify coherent, executable models composed from a collection of modules and provides numerous options for advanced biomodel engineering.