Urokinase receptor in regulation of cellular senescence and DNA damage response: role for the ubiquitin-proteasomal system Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktorin der Naturwissenschaften Dr.rer.nat genehmigte Dissertation von Mahshid Hodjat, Master geboren am 05.09.1978, in Shemiran Hannover, 2012 2013 Referent: Prof. Dr. rer. nat Inna Dumler Co-Referent: PD Dr. rer.nat Karsten Grote Tag der Promotion: 14 March 2013 II III The present study was carried out at the department of Nephrology, Vascular biology lab at Hannover Medical School Parts of this study have been published: Urokinase receptor mediates doxorubicin induced vascular smooth muscle cells senescence via proteasomal degradation of TRF2. Mahshid Hodjat, Hermann Haller, Inna Dumler, and Yulia Kiyan. Journal of vascular biology, 2013;50:109-123 Loss of urokinase receptor sensitizes cells to DNA damage and delays DNA repair .Yulia Kiyan, Mahshid Hodjat, Hermann Haller, and Inna Dumler Submitted at journal of cell science. 2012 Abstracts: uPAR regulates vascular smooth muscle cells senescence by interfering with Telomeric Repeat Binding Factor 2 (TRF2) proteasomal degradation. XlIIth international workshop on Molecular & Cellular Biology of Plasminogen Activator, July 9-13, 2011Clare College Cambridge, England uPAR mediates Telomeric Repeat Binding Factor 2 proteasomal degradation in senescence. Life in motion: Dynamics of Molecules and Systems. 2nd IRB Barcelona PhD Student Symposium. 17-18 November 2011 IV List of abbreviations ATM Ataxia-telangiectasia mutated BARD BRCA-associated ring domain bFGF Active fibroblast growth factor BRCA1 Breast cancer type BRIT1 BRCT-repeat inhibitor of hTERT expression cAbl Abelson murine leukemia viral homolog 1 Chk Checkpoint kinase CP Core particle DNA-PKcs DNA-dependent protein kinase catalytic subunit DOX Doxorubicin DSBs Double-strand breaks DSS1 Deleted in split hand/split foot protein 1 ECM Extracellular matrix EGF Epidermal growth factor EGFR Epidermal growth factor receptor ERK Extracellular signal-regulated kinase FANCL Fanconi anemia complementation group L FPRL Formyl peptide receptor-like GPCR G protein coupled receptor GPI Glycosylphosphatidylinositol HR Homologous recombination IRIF Ionizing radiation-induced foci JAK Janus kinase kDa Kilodaltons LRP Low density lipoprotein receptor-related protein mAb Monoclonal antibody MCPH1 Microcephalin MDa Megadalton Nbs1 Nijmegen breakage syndrome 1 Mdc1 Mediator of DNA-damage checkpoint 1 Mdm2 Murine double minute MFI Mean fluorescence intensity MMP Matrix metalloproteinase MRN Mre11-Rad50-Nbs1 NF-κB Nuclear factor-kappaB NHEJ Nonhomologous end joining 53BP1 p53 binding-protein 1 pAb Polyclonal antibody PAC Proteasome assembly chaperones PAI-1 Plasminogen activator inhibitor type-1 PAI-2 Plasminogen activator inhibitor type-2 PAS Plasminogen activation system V PDGFRß Platelet-derived growth factor receptor beta PIKK Phosphoinositide-3-kinase-related protein kinase PKC Protein kinase C POT1 Protection of Telomeres 1 Pro-MMPs Pro-metalloproteinases Pro-uPA Pro-urokinase RP Regulatory particle SAβG Senescence-associated β-galactosidase SHFM1 Split hand-split foot malformation 1 SIAH Seven in absentia homolog siRNA Small interfering RNA SS Single-stranded STAT Signal Transducer and Activator of Transcription suPAR Soluble urokinase-type plasminogen activator TIN2 TERF1-interacting nuclear factor2 TGF-β Transforming growth factor-beta TopBP1 Topoisomerase IIβ-binding protein 1 TPP1 TEL patch of telomere protein 1 TRF1 Telomeric-repeat binding factor1 UBC Ubiquitin-conjugating enzyme UBE1L2 Ubiquitin-activating Enzyme 1-like 2 UPS Ubiquitin-proteasome system uPA Urokinase-type plasminogen activator uPAR Urokinase-type plasminogen activator receptor VEGF Vascular endothelial growth factor VSMC Vascular smooth muscle cell VI Table of contents List of abbreviations .................................................................................................................. V Table of contents ..................................................................................................................... VII Abstract .................................................................................................................................... IX Zusammenfassung ..................................................................................................................... X 1.Introduction ............................................................................................................................. 1 1.1.Urokinase type plasminogen activator receptor ............................................................... 2 1.1.2. uPAR structure ......................................................................................................... 2 1.1.3. uPAR expression ...................................................................................................... 3 1.1.4. uPAR functions ........................................................................................................ 4 1.1.4.1. Proteolytic functions .............................................................................................. 4 1.1.4.2. Non-proteolytic functions ...................................................................................... 4 1.1.5. Role of uPAR in vasculature and vascular diseases ................................................. 7 1.2. DNA damage response .................................................................................................... 8 1.2.1. Telomere and DNA damage response .................................................................... 11 1.2.2. Telomere binding proteins and their function ........................................................ 11 1.2.3. The DNA damage hypothesis of telomere dysfunction ......................................... 13 1.2.4. Senescence as a DNA damage response ................................................................ 13 1.2.5. Cellular senescence implication in vasculature ...................................................... 14 1.3. Ubiquitin-proteasome system (UPS) ............................................................................. 15 1.3.1. Ubiquitination mediating enzymes ......................................................................... 15 1.3.2. 26S proteasome structure ....................................................................................... 16 1.3.3. Regulation of proteasomal activity ......................................................................... 16 1.3.4. Regulatory role of Ubiquitin-proteasome system in DNA damage response ........ 17 2. Objectives ............................................................................................................................. 19 3. Publications and manuscripts ............................................................................................... 22 3.1. Manuscript 1 .................................................................................................................. 39 3.2. Manuscript 2 .................................................................................................................. 40 4. Discussions ........................................................................................................................... 64 5. References ............................................................................................................................ 72 6. Supplementary data .............................................................................................................. 82 VII 7. Appendix .............................................................................................................................. 87 Table of figures: ................................................................................................................... 88 Acknowledgements .............................................................................................................. 89 Curriculum vitae ................................................................................................................... 91 VIII Abstract Abstract DNA damage is a relatively common event in the life of cell. Based on type and location of DNA lesion several cellular responses are induced that enables the cell either to repair the damage or to activate a programmed cell senescence or death process. The precise knowledge of the molecular mechanisms that determine DNA damage lesion and downstream signaling response remains, however, elusive. The uPA/uPAR is a multifaceted system mediating a diverse array of extracellular and intracellular processes. We observed that the level of uPAR expression plays a decisive role in regulation of telomeric repeat binding factor 2 (TRF2) during doxorubicin-induced senescence. TRF2 is the main telomere-associated protein implicated in the maintenance of DNA structure and known to be necessary for proper telomere function. Our results demonstrate that uPAR controls the ubiquitin-proteasome system in vascular smooth muscle cells (VSMC) and regulates doxorubicin-induced TRF2 ubiquitination and proteasomal degradation via this mechanism. Using repairable DNA damage response model, we found that uPAR also involved in DNA damage repair mechanisms. The underlying pathways involved uPAR-mediated regulation of the proteasome regulatory subunit, Rpn7 and its redistribution to DNA damage foci. We further showed