MARTIN-LUTHER- UNIVERSITÄT HALLE- WITTENBERG Medizinische Fakultät Julius-Bernstein-Institut für Physiologie Direktor: Prof. Dr. med. Michael Gekle Datum 25.08.2014 Promovieren als Student/Studentin der Medizin oder Zahnmedizin im DFG-Graduiertenkolleg 1591 Am Julius-Bernstein-Institut für Physiologie sind im Rahmen des DFG-geförderten Graduiertenkollegs DFG-GRK 1591 Posttranskriptionelle Regulation der Genexpression - Mechanismen und Rolle in der Pathogenese ab Oktober 2014 zwei Doktorandenstellen zu vergeben. Die Mitgliedschaft im Graduiertenkolleg bietet - Strukturierte Promotion - Intensive Betreuung und Beratung - Regelmäßige Seminare und Retreats - Großes Angebot an Zusatzveranstaltungen im theoretischen, praktischen und ASQ Bereich - Umfassende Einblicke in die biomedizinische Forschung - Regelmäßigen Austausch mit anderen DoktorandInnen auch aus anderen Fakultäten - Stipendien über 12 Monate oder länger - Finanzielle Unterstützung bei Kongressreisen Hausanschrift: Magdeburger Str. 6 06112 Halle/Saale Germany Tel. (0345) 557 1886 Fax (0345) 557 4019 Sekretariat: Kerstin Quarch Tel.: (0345) 557 1886 Fax: (0345) 557 4019 e-mail: kerstin.quarch@medizin.uni-halle.de Internet: www1.medizin.uni-halle.de/iph/ Direktor: Prof. Dr. Michael Gekle Tel.: (0345) 557 1886 Tel.: (0345) 557 1389 Fax: (0345) 557 4019 e-mail: michael.gekle@medizin.uni-halle.de Jeweils eine Doktorandenstelle ist in folgenden beiden Projekten zu vergeben: B1: Expression and function of the noncoding WISP1 transcript (lncwisp1) in fibrosis B2: The role of mir-221/222 in epidermal growth factor receptor (EGFR)-mediated changes in the cardiovascular system Ansprechpartner sind: Jun-Prof. Dr. Dr. Claudia Grossmann (Claudia.grossmann@medizin.unihalle.de) Dr. Barbara Schreier (barbara.schreier@medizin.uni-halle.de) PD Dr. Gerald Schwerdt (gerald.schwerdt@medizin.uni-halle.de) Prof. Dr. Michael Gekle (michael.gekle@medizin.uni-halle.de)
B1: Expression and function of the noncoding WISP1 transcript (lncwisp1) in fibrosis Michael Gekle, Gerald Schwerdt; Julius-Bernstein-Institute of Physiology, Medical Faculty, University of Halle-Wittenberg Specific aim(s)/topic(s) (1) Characterizing the functional principle of the long noncoding RNA lncwisp1 (2) Impact of lncwisp1 on cellular phenotype Background and significance Chronic interstitial dysregulation of tissue homeostasis (parainflammation) is a risk factor for fibrosis and carcinogenesis. The mechanisms of unbalanced tissue homeostasis involve altered transcriptional and posttranscriptional regulatory mechanisms, including noncoding RNAs as micrornas (mir) or long noncoding RNAs (lncrnas). LncRNAs belong to a rapidly growing class of noncoding RNAs and are defined as transcribed RNA molecules longer than 200 nucleotides [1]. They are involved in processes like chromatin modification, transcriptional and posttranscriptional gene regulation [2]. For example, lncrnas can compete with mir target genes for shared mirs (sponge-like action) leading to a dysregulation of the respective mir target genes. Wnt1 inducible signaling pathway protein 1 (WISP1) is described to be involved in organ development but also in fibrogenic and cancerous tissue alterations [3]. It belongs to the CCN family of connective tissue growth factors and is canonically regulated by the Wnt1/β-catenin pathway. Also, some splice variants of WISP1 with mostly unknown function have been described. Our recent data showed that WISP1 RNA is upregulated by different cell stressors in renal cells leading to increased formation of extracellular matrix components such as collagens which suggests a role of this RNA in pathological cellular processes [4]. The further identification of triggers and mechanisms (relevant cell stressors, signaling pathways involved in noncoding RNA expression, interaction of noncoding RNAs, crosstalk with target genes) involved in WISP1 RNAinduced dysregulation of tissue homeostasis may contribute to the understanding of organ and tissue remodelling and malfunction. Previous and preliminary studies In primary as well as in cultured human renal proximal tubule cells we characterized further the above mentioned WISP1 RNA and identified a so far not described alternative transcript of WISP1 which is markedly upregulated upon exposure to different cell stressors (here ochratoxin A, a fungal metabolite with harmful potential on renal cells [4], phorbol esters and NO-donors). In subsequent studies it turned out that this transcript very likely does not code for a protein (all antibodies tested failed to detect WISP1 protein, nuclear localisation as described below), hence it is a representative of the new class of long noncoding RNAs and we named it lncwisp1 (long noncoding WISP1). LncWISP1 is induced by different cell stressors in a MEK1/2-dependent pathway. Detailed analysis of lncwisp1 (including 3 - and 5 -RACE) revealed that it is a 2922 nucleotide-long so far unknown transcript which consists of only a part of the fifth exon includ-
ing the 3 UTR which contains mir target sites for mir-15, mir-23, mir-27, mir-214 and mir- 29b. We could demonstrate that lncwisp1 is located almost entirely to the nucleus (150-fold enrichment over cytosol) and that it induces a partial nuclear relocalisation of mir-29b. Stressor-induced upregulation of lncwisp1 resulted in enhanced collagen I and III mrna and protein expression, which was abolished by a mir-29b clamp using mimics. Thus, lncwisp1 possibly acts as a profibrotic regulator acting by disinhibition through its interference with at least mir29b and possibly other mirs. Preliminary observations suggest additional effects of lncwisp1: overexpression of lncwisp1 not only reduces the mir-29b-dependent luciferase reporter activity, but also that of a mir-449c luciferase reporter although no known seed region for mir-449c is predicted in lncwisp1. This indicates a new and possibly more generalised mode of action of lncwisp1 on mirs which must be further characterised. In addition we have some indications that lncwisp1 affects the level of pri-mir29b, suggesting a transcriptional effect. Research design and methods A. Characterization of lncwisp1 expression. To conclude the basic characterization of lncwisp1 its half-life will be determined in primary human renal cells (RPTEC) and different human renal and non-renal cells (HEK293, HK2, fibroblasts, cancer cells). Besides that, the lncwisp1 promotor will be characterised. It is presumable that the expression of lncwisp1 is under the control of an alternative promotor. Therefore, the identification of this promotor (by use of reporter construct) and of response elements therein as well as signalling pathways leading to the regulation of this alternative promotor will be investigated. In preliminary experiments we cloned the putative promotor and gained evidence that the promotor is located in intron 4. B. Characterization of lncwisp1 action. We will determine the functional interaction of lncwisp1 with mirs which are predicted to have or to have not seed regions in lncwisp1. Therefore, luciferase reporter plasmids containing the respective mir binding sites as well as mir overexpressing plasmids will be used and tested in competition assays with lncwisp1. The effect of wild type lncwisp1 overexpression will be compared to the effect of constructs with deleted or mutated putative mir binding sites (seed regions). Furthermore, we will apply the reverse approach using a lncwisp1-3 UTR-reporter (with wild-type or deleted mir seed regions) and test the effect of mir overexpression. To identify lncwisp1 interacting partners (mirs or proteins) the mir-trap system (cooperation with B3) will be used. In order to study the influence of lncwisp1 on nuclear-cytosolic mir distribution (as already shown for mir-29b) their compartimentation will be investigated on the basis of mirs with or without putative seed regions in lncwisp1. The paradigm of a transcriptional action of lncwisp1 will be tested using mir-29, for which the preliminary data indicate an effect on pri-mir-29 generation. We will use the actinomycin D approach, determine pri-mir half-life and invesitgate the mir-29 promotor. C. Cellular consequences of lncwisp1 expression. In a targeted approach the effect of lncwisp1 overexpression on cellular matrix homeostasis (collagen I, III, IV and fibronectin) will be determined. In a screening approach the effect of lncwisp1 overexpression on gene expression will be determined by disease-specific gene arrays (genes related to fibrosis or epithelial-tomesenchymal transformation) and subsequent validation. D. Determination of the occurrence of lncwisp1 in healthy and diseased tissues as well as in cells from healthy and diseased tissue. The amount of lncwisp1 expression in renal tumour and healthy tissues (cooperation with the P. Fornara, section of urology, University Halle) as well as in renal and non-renal cancer cell lines (cooperation with B4) will be determined.
B2: The role of mir-221/222 in epidermal growth factor receptor (EGFR)-mediated changes in the cardiovascular system B. Schreier, C. Grossmann; Institute of Physiology, Medical Faculty, University of Halle Specific aim(s)/topic(s) (1) the role of EGFR for cardiovascular mir-221/222 dysregulation (2) molecular mechanisms leading to EGFR-induced changes in mir-221/222 (3) cellular and functional consequences of EGFR-dependent mir-221/222 dysregulation Background and significance The EGFR is a ubiquitously expressed receptor tyrosine kinase that can be activated by different ligands like EGF but it can also be transactivated by a variety of stimuli including G-proteincoupled receptors and cellular stress, making the EGFR an important interconnection. In the cardiovascular system, an association of EGFR with impaired vascular and cardiac function and parainflammatory remodeling processes has been made based on clinical and experimental data of pharmacological EGFR inhibitors and mice with reduced EGFR tyrosine kinase activity [1]. The results are partially contradictory and the underlying molecular mechanisms are not completely understood. In the recent years small non-coding RNAs (mir) have been implicated in the regulation of pathological cardiac and vascular phenotype and the EGFR signaling cascade has been suggested as a regulator of mir expression [2-5]. One of the mir clusters associated with cardiovascular homeostasis and pathology is mir-221/222 [6-7]. These mirs also show altered expression in aorta and heart of mice with targeted deletion of EGFR, suggesting an involvement of mir-221/222 in EGFR-mediated effects in the cardiovascular system. Previous and preliminary studies During the past years, we have analyzed the role of the EGFR in cardiac and vascular function employing a mouse model with targeted deletion of the EGFR in cardiomyocytes and vascular smooth muscle cells (VSMC) using the SM22-promoter [8-9]. EGFR knockout mice showed a significant reduction in life span and suffered from a dramatic eccentric cardiac hypertrophy (2.5fold increase in heart weight) with increased stroke volume and left ventricular wall and lumen thickness. Cardiac hypertrophy was accompanied by an increase in cardiomyocyte volume and elevated markers of fibrosis and inflammation, as well as enhanced NOX4 expression, suggesting an impaired ROS homeostasis as trigger for these structural changes [9]. In the vasculature, there EGFR knockout resulted in no changes in systolic blood pressure whereas total peripheral vascular resistance, diastolic and mean blood pressure were reduced. A dilated vascular phenotype with minor signs of fibrosis and inflammation was detected. In isolated VSMCs, EGFR deletion affected ROS and matrix homeostasis as well as ERK1/2 and calcium signaling in response to EGFR transactivation, demonstrating the importance of EGFR for basal VSMC homeostasis [8]. We could also demonstrate the relevance of EGFR for random and targeted VSCM migration upon stimulation with EGFR specific ligands and for transactivation-induced migration by vasoactive peptides (unpublished data). To evaluate the underlying mechanism of EGFR-mediated cardiovascular effects, we analyzed RNA isolated from hearts and aortas and could detect and validate a significant upregulation of mir-221/-222 in hearts and/or aorta of knockout animals (aorta, mir-221: C t 1.5 ± 1.2, mir- 222: C t 0.7 ± 0.9; heart, mir-221: C t 1.1 ± 0.6, mir-222: C t 1.3 ± 0.6, mean ± 95% CI). Based on these results, we hypothesize that EGFR is an upstream regulator of the mir-221/222
cluster and that these mirs are involved in the pathophysiological role that the EGFR plays in the cardiovascular system. Research design and methods The role of EGFR for mir-221/222 dysregulation in the cardiovascular system Based on the observed upregulation of mir-221/222 in the heart and aorta of EGFR knockout mice, we plan to analyze mir-221/222 and pri-mir-221/222 expression in primary cardiomyocytes, cardiac fibroblasts and VSMC of our EGFR knockout mouse model by TaqMan qpcr and Northern Blot under basal conditions, after ligand stimulation (EGF) and after transactivation with vasoactive substances and different types of stress. If necessary we will take advantage of the pharmacological EGFR inhibitor AG1478 to verify the observed effects. These data will be confirmed in heart and aorta of our EGFR knockout mice by in vivo experiments. Molecular mechanisms leading to EGFR-induced changes in mir-221/222 To investigate the mechanism by which EGFR leads to changes in mir-221/222, we will measure mir-221/222 and pri-mir-221/222 in the presence and absence of actinomycin D and after dicer knockdown with sirna, discriminating between effects of EGFR on mir-transcription, stability and different processing steps. To validate the functionality of the changes in mir-221/222, a 3 - UTR reporter gene assay with the common seed region will be performed. Furthermore, we will inhibit different downstream signaling cascades of the EGFR (e.g. pakt, perk, Stat5, c-src, p38) by chemical inhibitors and survey mir-221/222 and pri-mir-221/222 by TaqMan qpcr to acquire information about the signaling molecules involved in EGFR-mediated regulation of mir- 221/222 expression. Cellular and functional consequences of EGFR-dependent mir-221/222 dysregulation The effect of EGFR-dependent mir-221/222 regulation will be tested under basal and stressinduced conditions with and without addition of antagomirs or mimics respectively a) in primary cardiomyocytes and VSMC of our EGFR knockout model. We will measure mrna and protein expression of target genes (Cdkn1b, KIT and exemplary genes identified in our previous publications) as well as cellular phenotype by evaluating cell death, fibrosis and inflammation and cellular function by measuring cell migration, cell contraction and excitation-contraction-coupling. b) in vivo in our EGFR knockout model. We will measure tissue homeostasis by evaluating organ size, histology and markers of inflammation, fibrosis and hypertrophy. Furthermore, organ function will be assessed by analyzing blood pressure (tailcuff and Millar catheter), aortic force generation, echocardiography and electrocardiography.