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Prof. Dr. rer. nat. Dr. med. habil. |
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Research Group KH. Friedrich What we do, where, how, and why ... Context Signal transduction is a central aspect of life. Well organized distribution of information throughout the human organism is crucial for its integrity and homeostasis. The body constitutes an extremely complex network of cells which release, perceive and process chemical as well as electrical signals. The work of our research group is concerned with molecular mechanisms of cellular signal transduction and with dysfunction of these processes in the course of disease. We strive for a better understanding of how cells and molecules "talk to each other" and try, by doing so, to contribute to novel developments in molecular medicine. People The photo from spring 2009, taken in the historical „Kollegienhof“, shows from left to right: Andreas Wohlmann, Nadine Knutti, Andreas Borowski („Boro“), Josi Gasch, Maria Tettenborn, Sebastian Krause, Frauke Zellmann, Andrey Bondev, Annekatrin Müller, Karlheinz Friedrich (missing: Susanne Grimm, Fritz Uthe).
If you are interested in a practical, in diploma or doctoral
work, sometimes we have opportunities. Just contact us under khf@mti.uni-jena.de! Place The laboratory is located within a medieval building complex (the historical center of the Friedrich Schiller University with its oldest parts built in the 16th century). In a somewhat bizarre fashion, this remarkable architectural ensemble called "Collegium Jenense" is contrasted by its immediate neighbourhood: The most striking object here is, without doubt, the "Intershop Tower", a highrise building from the nineteen-seventies overtopping the town center with its really interesting mixture of pittoresque and confusing sights by more than 100 m... The air view (taken from an ultralight airplane while cruising above the city center...) shows the overall situation, the other photographs are meant to give some impressions how it looks like around the institute. Jena, in any case, is a very agreable, communicative place for research, combining history with high tech, an inspiring cultural life, and a truely marvellous natural environment.
Projects 1. Cytokine signaling in allergic diseases: Roles of Interleukin-13 and Thymic Stromal Lymphopoietin (TSLP) in Asthma bronchiale Interleukin-13 (IL 13) is a cytokine with multiple functions in normal immune processes and inflammatory diseases. It is mainly produced by activated T-cells, but also by various other cell types such as mast cells, basophils, eosinophils and dendritic cells. Although IL-13 is structurally and functionally closely related to IL-4, it plays a specific critical role in the development of allergic asthma, a chronic disease of the airways associated with hyperresponsiveness to specific antigens, elevated IgE levels, mucus hypersecretion and subepithelial fibrosis.
The IL 13 receptor (IL-13R) is a typical bipartite cytokine receptor which is activated by ligand-induced dimerization and signals through Janus kinases and Signal Transducers and Activators of Transcription (STATs). The two components of the IL-13R system are the IL-13Ra1 chain and the IL-4Ra chain (the latter is also a component of the IL-4 receptor). Importantly, the IL-13R is not exclusively utilized by IL-13, but can also be activated by IL-4. It, thus, constitutes also a "type II" IL-4 receptor in addition to the type I IL-4 receptor made up of a heterodimer of IL-4Ra with the common g receptor chain (gc). These overlapping receptor systems explain the close functional relationship of IL-13 and IL-4.
Research by our group in the 1990s on the IL-4R complex paved the way for our recent research on the IL-13R system. We reconstituted functional receptor complex by stable transfection in murine pro-B cells and performed an extended mutational analysis of receptor components and intracellular signal mediators. Results of these investigations were for instance the identification of STAT5 as an additional IL-4R signal transducer and the surprising finding that homodimers of the IL-4Ra hain are competent of intracellular signal transmission. Ways of specific experimental manipulation of IL-4-induced signaling reactions have been devised, e.g. by the use of dominant-negative signal mediator domains.
The current project aims at a better molecular understanding of specific IL-13 receptor function, IL-13-driven signal transduction and the influence of IL-13 on acute and chronic inflammation of the airways in the course of asthma. Finally, we put much effort in the development of agents that can specifically inhibit IL-13 receptor activation and, thus, may provide novel perspectives for the treatment of asthma symptoms.
We could show in the last years that interleukin-4- and interleukin-13 receptors trigger distinct intracellular signaling events and that the sensitivity of asthma-relevant eosinophilic granulocytes towards IL-13 is probably regulated through expression control of the IL-13 receptor a subunit. Because of the central role of IL-13 in asthma, the IL-13 receptor system is an obvious pharmaceutical target. Importantly, we succeeded recently in generating monoclonal antibodies to the IL-13Ra1 that can efficiently and specifically block the stimulation of cells by IL-13 without affecting their response to IL-4 (see figure). In this collaborative effort (together with the group of J.C. Virchow in Rostock and with Genovac AG in Freiburg), genetic immunization of mice was combined with the application of a novel cell-based read-out assay for the screening and characterization of hybridoma clones. The best antibodies obtained inhibit activation of the human IL-13-dependent cell proliferation and gene activation in both cell lines and primary cells from human blood with inhibition constants in the nanomolar range. The figure shows dose-dependent inhibition of IL-13- but not IL-4-dependent tyrosine phosphorylation in monocytes from human blood.
Presently, we exploit the potential of blocking the IL-13 receptor to ameliorate asthma symptoms. To approach the actual disease situation as closely as possible, we have started to use an ex vivo setting and investigate IL-13-induced activation and cytokine production of immune cells obtained from the airways of healthy humans and asthma patients. Furthermore, we study in cell culture models the role of IL-13 signaling in chronic inflammation of the lung, mainly in the development of fibrosis (replacement of functional lung epithelium by fibroblasts) as a long term consequence of asthma. Recently, we embarked on a new project centered around Thymic Stromal Lymphopoietin (TSLP). This interleukin-7-like cytokine emerged in the last years as a central player in the development of allergic symptoms, especially in the airways, and is a prime regulatory cytokine at the interface of virus- or antigen-exposed epithelial cells and dendritic cells. Since TSLP links contact of allergen with the airway epithelium to the onset and maintainance of the asthmatic syndrome, defining the signal transduction underlying TSLP expression and function is of profound interest for a better understandimg of the disease and for the development of new therapeutics. We have functionally reconstituted the TSLP receptor (TSLPR), a heterodimer of the IL-7R a-chain and a specific TSLP receptor subunit in model cell lines. Currently we study TSLP-driven JAK/STAT signalling and expression regulation of TSLP target genes. Moreover, we attempt to generate antagonistic antibodies to the TSLPR with the ultimate goal of developing novel inhibitors for TSLP activity in asthma. Literature
2. Development of novel therapeutical activators and inhibitors of cytokine/growth factor receptors The activity of signaling molecules on responder cells via transmembrane receptors is crucial for the coordination throughout the organism. Inadequate function of the underlying processes causes or accompagnies numerous disease states including immune disorders and cancer. Receptors for growth factors and cytokines, thus, constitute attractive targets for pharmaceutical interference. In the 90s, we were concerned with the development of assays that facilitate the detection and biochemical characterization of interactions between soluble ligands and membrane-bound receptors. The intention behind this research was to open up better ways for the targeted manipulation of clinically and pharmaceutically relevant receptor systems. Recently, we have developed a cell-based versatile and automatable functional test system for different receptors that become dimerized upon ligand binding. Now we employ it for the identification and characterization of molecules that interfere with various cytokines and growth factors of medical importance. Hybrid receptors were constructed from the cytoplasmic domains of the human interleukin-4 (IL-4) receptor and extracellular domains of representatives from different dimerizing, receptor systems (including the IL-13R described in Project 1, the receptors for erythropoietin and thrombopoietin and the receptor for TGF- (Transforming Growth Factor-) b). These chimeras were introduced into the factor-dependent murine cell line Ba/F3 along with an IL-4-inducible luciferase reporter gene construct. Reporter cell lines were obtained that respond to productive interactions of ligands with the respective receptor ectodomains by specific luciferase activity in a dose-dependent manner. The figure depicts the general experimental outline (left) and an experiment showing the specific effect of an antagonistic peptide on the response on engineered TGF-b reporter cells.
A recent outcome of our research was the identifiction of novel agonists of the thrombopoietin receptor. Thrombopoietin (TPO) is the primary regulator of platelet production. Low platelet count (Thrombocytopenia) is a serious medical problem for instance in the course of cancer chemotherapy. The application of recombinant TPO has faced the problem that the recipients frequently develop neutralizing antibodies. TPO-mimetic peptides, on the other hand, are too labile for the utilization as drugs. In collaboration with the group of H. Kolmar in Göttingen/Darmstadt, we have exploited cystine knot miniproteins as a starting point for the engineering of novel TPO mimetics. Using TPO reporter cells, we could identify and characterize a miniprotein-derived TPO agonist with a biological activity comparable to natural TPO. It holds considerable potential with regard to the future development of stable and immunulogically inert pharmaceuticals for the treatment of thrombocytopenia. An intriguing perspective for us is the development of protocols that allow for the direct selection of functional cytokine/growth factor mimetics from molecular repertoires and display libraries. One step in this direction was the demonstration that cytokines can be displayed on the surface of bacteria such that they specifically stimulate mammalian cells. Literature
3. Pathological JAK/STAT signaling in cancer Signal Signal transduction through Janus kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs) is essential for a multitude of fundamental cell functions such as proliferations and differentiation. Best characterized is the triggering of JAK/STAT signaling by cytokine receptors. JAKs, upon receptor dimerization, phosphorylate the receptor and, thus, generate docking sites for signaling proteins comprising SH-2 domains, e.g. STATs. In the course of interaction with the cytokine receptor complex, STATs themselves become tyrosine phosphorylated. Subsequently, they dimerize, translocate to the cell nucleus and, upon binding to cognate DNA elements, act as transcriptional modulators of cytokine target genes. Because of of their central importance for the cell fate it is not surprising that aberrant activation of JAK/STAT pathways has been observed in various human cancers. Our group has a long standing interest in the molecular mechanisms of signal transduction from cytokine receptors to target genes via JAKs and STATs and in resulting cellular responses. In recent years, we focussed our attention on the role of dysregulated JAK/STAT signaling in different types of malignant human tumors, namely colorectum and lung cancer and chorion carcinoma. We ask in how far aberrant STAT activity is operative in cancer tissue, if it is of importance for the malignancy of the tumor, and what signaling mechanisms functionally connect the activities of STATs and other molecules with pathogenetic properties. To address these problems, we study cancer biopsies and related cancer cell lines in a comparative fashion. These investigations are performed in collaboration with several other laboratories, among them the groups of Richard Moriggl (Vienna), Stephan M. Feller (Oxford) and Alexander Pupyshev (Novosibirsk). Colorectal cancer (CRC) and lung cancer rank among the tumor diseases with the highest incidence and death rates in the “developed” countries. We observed constitutive tyrosine phosphorylation and DNA binding activity of STAT3 in biopsies from the majority of malignant colorectal and lung tumors and the specific localization of tyrosine phosphorylated STAT3 in the nuclei of carcinoma cells (see figure). Studies on various tumor cell lines transfected with different STAT3 variants revealed that STAT3 activity directly drives cell proliferation and invasiveness. In xenogaft experiments, these cell lines formed tumors upon implantation into immunodeficient mice. Most importantly, inhibition of STAT3 activation by expression of a dominant-negative mutant resulted in a significant reduction of tumor growth. These results indicate that STAT3 is immediately associated with the pathogenesis of solid cancers such as CRC and constitutes an attractive target for therapeutic intervention.
Another issue of the project is the investigation of STAT3-dependent alteration of gene expression patterns in the course of oncogenic transformation of colon and lung epithelial cells. We have used cDNA arrays covering protease sequences and found an association of STAT3 activity with elevated expression of matrix metalloproteinases (MMPs). Reporter gene assays and promoter studies unraveled a direct functional connection between STAT3-driven gene regulation and MMP expression. Current work is concerned with molecular analysis of transcriptional regulation of the MMP-1 promoter by STAT3. A particularly important aspect of tumor malignancy is the ability of tumor cells to grow in an invasive manner and to ultimately form metastases at distant locations in the organism. STAT3 is an important determinant of tumor-stroma interactions, of cellular invasiveness and, probably, matastasis. We are, thus, studying the contribution of STAT3 to the invasive behavior of tumor cells in vitro and in vivo. Moreover, we have obtained evidence for correlations between the grade of STAT3 activation and the tendency of primary tumors to form metastases. Literature
4. A novel gene therapeutic approach to study and block the activity of oncogenic Ras Ras proteins play an important role in receptor-mediated signal transduction pathways. They control cell proliferation and differentiation and are involved in cell motility and invasiveness. As G-proteins, Ras and its relatives cycle between an active GTP-bound and an inactive GDP-bound form. Oncogenic mutations that compromise the intrinsic and GAP-promoted GTPase activity of Ras proteins have been found in up to 30% of human tumors. Due to its involvement in malignant cell transformation, Ras is an attractive target for therapeutic intervention. In a colloaborative project with the group of Ignacio Rubio (Jena), we have developed the Multivalent Scavengers of Oncogenic Ras (MSOR) concept as a novel approach to interfere with aberrant Ras activity. MSORs consist of oligomerized c-Raf Ras-binding domains (RBD) or mutants thereof. Oligomerization of RBDs resulted in a particularly efficient interaction with Ras owing to the multivalent binding to Ras-GTP. MSORs proved able to specifically target active, GTP-bound Ras, thereby blocking Ras-mediated signalling and cell transformation as examplified by ERK1/2 phosphorylation, specific gene transcription, anchorage-independend and invasive growth. GFP-tagged MSORs enabled us to monitor EGF-induced spatio-temporal Ras activation in live cells. The figure depicts the general concept and the dependency of cellular effects to MSORs (apoptosis and colony formation in a soft agar assay) on the grade of oligomerization.
The strength of Ras blockade could be adjusted by using MSORs derived from various mutant RBDs with different affinities for Ras. Moreover, experimental alteration of MSOR expression by means of a regulatable promoter rendered Ras-dependent malignant cell transformation reversible in a graded and inducible fashion. Current efforts aim at demonstrating that MSORs can ameliorate Ras-driven tumor growth in animals. Literature
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