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Neue Regulatorische Effekte Von GLI2 Und HHAT Auf Die Aktivierung Der Hedgehog Signalkaskade in Der Systemischen Sklerose Und Ch

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Neue Regulatorische Effekte Von GLI2 Und HHAT Auf Die Aktivierung Der Hedgehog Signalkaskade in Der Systemischen Sklerose Und Ch Neue regulatorische Effekte von GLI2 und HHAT auf die Aktivierung der Hedgehog Signalkaskade in der systemischen Sklerose und Charakterisierung der Rolle des Orphan Nuclear Receptors RORα in der Pathogenese fibrotischer Erkrankungen Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Ruifang Liang 2019 Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 13th Dec 2019 Vorsitzender des Promotionsorgans: Prof. Dr. Georg Kreimer Gutachter/in: Prof. Dr. rer. nat. Michael Stürzl Prof. Dr. Steffen Backert Novel Insights into Hedgehog Signaling in Systemic Sclerosis at the level of GLI2 and HHAT and Characterization of the Role of the Orphan Nuclear Receptor RORα in Fibroblast Activation and Tissue Fibrosis To the Faculty of Natural Sciences of Friedrich-Alexander-University Erlangen-Nuremberg for the obtainment of the academic degree doctor rerum naturalium (Dr. rer. nat.) submitted by Ruifang Liang 2019 Approved by the Faculty of Natural Sciences of Friedrich-Alexander-University Erlangen-Nuremberg Date of oral examination: 13th Dec 2019 Chairman of examination board: Prof. Dr. Georg Kreimer Referee: Prof. Dr. rer. nat. Michael Stürzl Prof. Dr. Steffen Backert Index of contents Zusammenfassung ............................................................................................... 1 Summary .............................................................................................................. 3 1 Introduction .................................................................................................. 5 1.1 Systemic sclerosis ....................................................................................................... 5 1.1.1 Clinical overview (Epidemiology and classification)...................................................... 5 1.1.2 Pathogenesis .................................................................................................................... 6 1.2 TGF signaling – key mediator of fibrosis ............................................................ 14 1.2.1 TGF superfamily ......................................................................................................... 15 1.2.2 TGF intracellular signaling ......................................................................................... 15 1.2.3 TGF signaling in fibrotic responses ............................................................................ 16 1.3 Hedgehog pathway .................................................................................................. 17 1.3.1 Canonical Hedgehog signaling ...................................................................................... 18 1.3.2 Non-Canonical Hedgehog signaling .............................................................................. 19 1.3.3 Hedgehog in fibrosis and cross talk with TGFβ signaling ............................................ 20 1.3.4 GLI proteins .................................................................................................................. 20 1.3.5 Skinny hedgehog ........................................................................................................... 21 1.4 Nuclear receptors ..................................................................................................... 22 1.4.1 ROR family ................................................................................................................... 25 1.4.2 Physiological Functions of RORα ................................................................................. 27 1.5 Animal models of Systemic sclerosis ...................................................................... 30 1.5.1 Bleomycin-induced skin fibrosis ................................................................................... 30 1.5.2 Fibrosis induced by overexpression of a constitutively active TGFβ receptor type 1 (TBRIact) ...................................................................................................................................... 31 1.5.3 DNA Topoisomerase I-induced fibrosis ........................................................................ 31 1.5.4 Bleomycin-induced pulmonary fibrosis ........................................................................ 32 1.6 Aims of the study ..................................................................................................... 33 2 Materials and Methods .............................................................................. 35 2.1 Material .................................................................................................................... 35 2.1.1 Chemicals ...................................................................................................................... 35 2.1.2 Auxiliary materials ........................................................................................................ 39 2.1.3 Instruments .................................................................................................................... 40 2.1.4 Commercially available systems (kits) .......................................................................... 42 2.1.5 Antibodies ..................................................................................................................... 43 2.1.6 Primers ........................................................................................................................... 44 2.1.7 Media, buffers and solutions ......................................................................................... 47 2.2 Methods .................................................................................................................... 51 2.2.1 Human skin biopsies...................................................................................................... 51 2.2.2 Cell culture .................................................................................................................... 51 2.2.3 Nucleofection ................................................................................................................ 53 2.2.4 FuGENE HD Plasmids Transfection ............................................................................. 53 2.2.5 Plasmids ......................................................................................................................... 54 2.2.6 Plasmid isolation (Maxi Prep) ....................................................................................... 54 2.2.7 Side-directed mutagenesis ............................................................................................. 55 2.2.8 Chromatin immunoprecipitation ................................................................................... 55 2.2.9 Luciferase-repoter assay ............................................................................................... 56 2.2.10 Coculture systems .......................................................................................................... 56 2.2.11 Mice ............................................................................................................................... 57 2.2.12 RNA Analysis ................................................................................................................ 60 2.2.13 Histological Analysis..................................................................................................... 60 2.2.14 Protein analysis .............................................................................................................. 61 2.2.15 Amplification and purification of adenoviral vectors .................................................... 64 2.2.16 Statistical Analysis ........................................................................................................ 65 3 Results .......................................................................................................... 66 3.1 Evaluation of the HH transcription factor GLI2 in SSc ...................................... 66 3.1.1 Upregulated expression of GLI2 in fibrotic conditions ................................................. 66 3.1.2 GLI2 is upregulated by TGFβ in a Smad-dependent manner ........................................ 67 3.1.3 GLI2 regulates TGFβ induced fibroblast activation ...................................................... 73 3.1.4 Fibroblast-specific knockout of Gli2 prevents TBRact-induced fibrosis ...................... 75 3.1.5 Pharmacological inhibition of GLI2 inhibits TGF-β-dependent fibroblast activation .. 77 3.1.6 Pharmacological inhibition of Gli-2 ameliorates TBRact -induced fibrosis ................. 78 3.1.7 Inactivation of Gli2 induces regression of pre-established bleomycin-induced pulmonary fibrosis 79 3.2 The Hedgehog aceyltransferase Hhat regulates canonical TGFβ-dependent fibroblast activation in SSc ................................................................................................ 80 3.2.1 HHAT is upregulated in activated fibroblasts in fibrotic skin ....................................... 80 3.2.2 HHAT expression is induced by canonical TGF signaling ......................................... 84 3.2.3 HHAT promotes TGF-dependent fibroblast-to-myofibroblast-differentiation ........... 86 3.2.4 Knockdown of Hhat ameliorates experimental fibrosis ................................................ 89 3.3 The orphan Nuclear
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