Heike M. Hermanns

Principles of interleukin (IL)-6-type cytokine signalling and its regulation.

The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), ciliary neurotrophic factor, cardiotrophin-1 and cardiotrophin-like cytokine are an important family of mediators involved in the regulation of the acute-phase response to injury and infection. Besides their functions in inflammation and the immune response, these cytokines play also a crucial role in haematopoiesis, liver and neuronal regeneration, embryonal development and fertility. Dysregulation of IL-6-type cytokine signalling contributes to the onset and maintenance of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis, multiple sclerosis and various types of cancer (e.g. multiple myeloma and prostate cancer). IL-6-type cytokines exert their action via the signal transducers gp (glycoprotein) 130, LIF receptor and OSM receptor leading to the activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) cascades. This review focuses on recent progress in the understanding of the molecular mechanisms of IL-6-type cytokine signal transduction. Emphasis is put on the termination and modulation of the JAK/STAT signalling pathway mediated by tyrosine phosphatases, the SOCS (suppressor of cytokine signalling) feedback inhibitors and PIAS (protein inhibitor of activated STAT) proteins. Also the cross-talk between the JAK/STAT pathway with other signalling cascades is discussed.

Plasticity and cross-talk of Interleukin 6-type cytokines

Interleukin (IL)-6-type cytokines are critically involved in health and disease. The duration and strength of IL-6-type cytokine-mediated signaling is tightly regulated to avoid overshooting activities. Here, molecular mechanisms of inter-familiar cytokine cross-talk are reviewed which regulate dynamics and strength of IL-6 signal transduction. Both plasticity and cytokine cross-talk are significantly involved in pro- and anti-inflammatory/regenerative properties of IL-6-type cytokines. Furthermore, we focus on IL-6-type cytokine/cytokine receptor plasticity and cross-talk exemplified by the recently identified composite cytokines IL-30/IL-6R and IL-35, the first inter-familiar IL-6/IL-12 family member. The complete understanding of the intra- and extracellular cytokine networks will aid to develop novel tailor-made therapeutic strategies with reduced side effects.

Novel Role of Janus Kinase 1 in the Regulation of Oncostatin M Receptor Surface Expression

The oncostatin M receptor (OSMR) is part of a heterodimeric receptor complex that mediates signal transduction of the pleiotropic cytokine OSM via a signaling pathway involving Janus kinases (Jaks) and transcription factors of the signal transducers and activators of transcription (STAT) family. Upon heterologous expression of the OSMR in several cell lines, we observed that its surface expression was significantly enhanced by coexpression of the Janus kinases Jak1, Jak2, and Tyk2 but not Jak3. Chimeric receptors consisting of the extracellular region of the interleukin-5 receptor β chain and the transmembrane and intracellular part of the OSMR were similarly up-regulated on the plasma membrane when Jak1 was coexpressed. The overall expression level of these constructs did not change significantly, but Jak1 coexpression increased the amount of endoglycosidase H-resistant, fully processed OSMR chimeras. Using mutated receptor and Jak1 constructs, we were able to demonstrate that association of Jak1 with the membrane proximal region of the receptor, but not its kinase activity, is necessary for this effect. Moreover, deletion of the OSMR box1/2 region also resulted in an improved surface expression indicating that this region may contain a signal preventing efficient receptor surface expression in the absence of associated Jaks. Finally we demonstrate that in Jak1-deficient cells, the endogenous OSMR is significantly down-regulated, an effect that can be reversed by transient expression of Jak1 in these cells.

Characterization of the signaling capacities of the novel gp130-like cytokine receptor.

The gp130-like receptor (GPL) is a recently cloned member of the family of type I cytokine receptors. The name reflects its close relationship to gp130, the common receptor subunit of the interleukin (IL)-6-type cytokines. Indeed, the recently proposed ligand for GPL, IL-31, is closely related to the IL-6-type cytokines oncostatin M, leukemia inhibitory factor, and cardiotrophin-1. The second signal transducing receptor for IL-31 seems to be the oncostatin M receptor β (OSMRβ). The present study characterizes in depth the molecular mechanisms underlying GPL-mediated signal transduction. GPL is a strong activator of STAT3 and STAT5, whereas STAT1 is only marginally tyrosine-phosphorylated. We identify tyrosine residues 652 and 721 in the cytoplasmic region of the longest isoform of GPL (GPL745) as the major STAT5- and STAT3-activating sites, respectively. Additionally, we demonstrate Jak1 binding to GPL and its activation in heteromeric complexes with the OSMRβ but also in a homomeric receptor complex. Most interesting, unlike OSMRβ and gp130, GPL is insufficient to mediate ERK1/2 phosphorylation. We propose that this is due to a lack of recruitment of the tyrosine phosphatase SHP-2 or the adaptor protein Shc to the cytoplasmic domain of GPL.

Cross-regulation of cytokine signalling: Pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation

The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1β, TNFα or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.

Non-redundant signal transduction of interleukin-6-type cytokines. The adapter protein Shc is specifically recruited to rhe oncostatin M receptor.

The common use of the cytokine receptor gp130 has served as an explanation for the extremely redundant biological activities exerted by interleukin (IL)-6-type cytokines. Indeed, hardly any differences in signal transduction initiated by these cytokines are known. In the present study, we demonstrate that oncostatin M (OSM), but not IL-6 or leukemia inhibitory factor, induces tyrosine phosphorylation of the Shc isoforms p52 and p66 and their association with Grb2. Concomitantly, OSM turns out to be a stronger activator of ERK1/2 MAPKs. Shc is recruited to the OSM receptor (OSMR), but not to gp130. Binding involves Tyr861 of the OSMR, located within a consensus binding sequence for the Shc PTB domain. Moreover, Tyr861 is essential for activation of ERK1/2 and for full activation of the α2-macroglobulin promoter, but not for an exclusively STAT-responsive promoter. This study therefore provides evidence for qualitative differential signaling mechanisms exerted by IL-6-type cytokines.

Signal transduction of IL-6, leukemia-inhibitory factor, and oncostatin M: structural receptor requirements for signal attenuation.

Stimulation of the IL-6R complex leads to Src homology domain containing tyrosine phosphatase 2 (SHP2) recruitment to the receptor subunit gp130 and its subsequent tyrosine phosphorylation. SHP2 is a two-SH2 domain-containing protein tyrosine phosphatase that is activated by many cytokines and growth factors. SHP2 counteracts the activation of transcription factors of the STAT family and the induction of IL-6-responsive genes. Tyrosine 759 of gp130, the signal transducing subunit of the IL-6R complex, is essential for the phosphorylation of SHP2. Mutation of tyrosine 759 to phenylalanine leads to an enhanced inducibility of IL-6-dependent genes. Here we demonstrate that no further tyrosines in the cytoplasmic part of gp130 are required for the phosphorylation of SHP2. We also tested whether the tyrosine 759 motifs in both subunits of the gp130 dimer are required for SHP2 association and tyrosine phosphorylation. Interestingly, one SHP2-recruiting phosphotyrosine motif in a single chain of the gp130 dimer is sufficient to mediate SHP2 association to the gp130 receptor subunit and its tyrosine phosphorylation as well as to attenuate IL-6-dependent gene induction. Furthermore, we show that repression of gene induction via Y759 does not require the presence of the SHP2 and STAT recruitment sites within the same receptor subunit, but within the same receptor complex. The Y759 motif in gp130 also attenuates gene induction mediated by the oncostatin M and leukemia inhibitory factor receptor complexes, which both contain gp130 as the shared subunit.

Oncostatin M and interleukin-31: Cytokines, receptors, signal transduction and physiology

Oncostatin M (OSM) and interleukin-31 (IL-31) are two cytokines belonging to the IL-6 family which share a common signaling receptor subunit, the OSM receptor beta (OSMRβ). Both of them are released by monocytes/macrophages, dendritic cells and T lymphocytes in inflammatory situations and upon binding to their respective receptor complexes they signal mainly via the JAK/STAT pathway. Besides sharing many biochemical properties, both display divergent physiological functions. This review summarizes aspects of cytokine transcription and biosynthesis, cytokine-receptor interactions, cross-species activities, signal transduction and physiology delineated from recent findings in genetic mouse models for both cytokines, OSM and IL-31.

Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis

OBJECTIVE To investigate the molecular mechanisms of CCL13/monocyte chemoattractant protein 4 (MCP-4) chemokine expression through proinflammatory cytokines in different primary human fibroblasts and the contribution of CCL13 to monocyte migration. METHODS Using RNase protection assays and enzyme-linked immunosorbent assays, we quantified the expression of CCL13 compared with that of CCL2/MCP-1 in primary human fibroblasts. Boyden chamber assays were performed to determine the importance of CCL13 for migration of primary monocytes. Pharmacologic inhibitors as well as small interfering RNA knockdown approaches were used to investigate the signaling pathways regulating CCL13 expression. RESULTS The interleukin-6 (IL-6)-type cytokine oncostatin M (OSM) was a powerful inducer of CCL13 expression in primary synovial fibroblasts from patients with rheumatoid arthritis (RA) as well as those from healthy control subjects but not in other types of fibroblasts. Neither IL-6 nor tumor necrosis factor alpha could stimulate the expression of CCL13 in synovial fibroblasts; IL-1beta was a very weak inducer. Synovial fibroblasts from patients with RA constitutively produced low amounts of CCL13, which was partially dependent on constitutive production of OSM. By investigating the underlying molecular mechanism, we identified STAT-5, ERK-1/2, and p38 as critical factors involved in OSM-dependent transcription and messenger RNA stabilization of CCL13. CONCLUSION In contrast to other prominent cytokines involved in the pathogenesis of RA, OSM can strongly up-regulate the expression of CCL13, a chemokine recently identified in the synovial fluid of patients with RA. Despite potent OSM-induced signal transduction in all types of fibroblasts analyzed, only synovial fibroblasts secreted CCL13, which might be indicative of tissue-specific imprinting of different fibroblasts during development.

A SINGLE STAT RECRUITMENT MODULE IN A CHIMERIC CYTOKINE RECEPTOR COMPLEX IS SUFFICIENT FOR STAT ACTIVATION

We established a system of receptor chimeras that enabled us to induce heterodimerization of different cytoplasmic tails. Fusion constructs were created that are composed of the extracellular parts of the interleukin-5 receptor α and β chains, respectively, and the transmembrane and intracellular parts of gp130, the signal transducing chain of the interleukin-6 receptor complex. In COS-7 transfectants we observed a dose-dependent interleukin-5-inducible STAT1 activation for which the presence of both the α and the β chain chimera was needed. No STAT activity was detected if one of the cytoplasmic tails of the receptor complex was deleted, indicating that STAT activity resulted from a receptor dimer rather than from higher receptor aggregates. We further investigated whether dimerization of STAT1 depends on the juxtaposition of two STAT recruitment modules in a receptor complex. We show that a receptor dimer with only a single STAT1 docking site was still able to lead to STAT1 activation. This indicates that the formation of a paired set of STAT binding sites in a receptor complex is not the prerequisite for STAT factor dimerization. Our findings are discussed in view of alternative STAT dimerization models.