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New Insights Into Signaling Mechanisms Controlling the Function and Fate of Hepatic Stellate Cells

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New Insights Into Signaling Mechanisms Controlling the Function and Fate of Hepatic Stellate Cells New insights into signaling mechanisms controlling the function and fate of hepatic stellate cells Dissertation This dissertation is submitted for the degree of the Doctor of Philosophy to the Faculty of Mathematics and Natural Sciences at the Heinrich‐Heine‐University Düsseldorf Presented by Saeideh Nakhaeirad from Mashhad, Iran Düsseldorf, November 2015 Neue Erkenntnisse über die Signalmechanismen in der Kontrolle der Funktion und des Schicksals hepatischer Sternzellen Inaugural‐Dissertation zur Erlangung des Doktorgrades der Mathematisch‐Naturwissenschaftlichen Fakultät der Heinrich‐Heine‐Universität Düsseldorf vorgelegt von Saeideh Nakhaeirad aus Mashhad, Iran Düsseldorf, November 2015 Aus dem Institut für Biochemie und Molekularbiologie II der Heinrich‐Heine‐Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch‐Naturwissenschaftlichen Fakultät der Heinrich‐Heine‐Universität Düsseldorf Referent: Prof. Dr. Reza Ahmadian Korreferent: Prof. Dr. Vlada Urlacher Tag der mündlichen Prüfung: 25 May 2016 Table of contents Summary ........................................................................................................................................ I Zusammenfassung ......................................................................................................................... II Table of Figures ............................................................................................................................ III Abbreviation ................................................................................................................................. IV Chapter I ........................................................................................................................................ 1 General introduction ................................................................................................................. 1 Anatomical units and resident cells of the liver .................................................................... 2 Microanatomy of the liver ................................................................................................. 2 Hallmarks and roles of hepatic stellate cells ......................................................................... 3 Quiescent HSC (qHSC) in the normal liver ......................................................................... 3 Activated HSC (aHSC) after liver injury .............................................................................. 6 Postulated signaling mechanisms in hepatic stellate cells .................................................... 7 RAS superfamily at a glance .............................................................................................. 8 RAS family GTPases ......................................................................................................... 10 Effectors and signaling of RAS proteins ........................................................................... 13 RHO family GTPases ........................................................................................................ 19 Signaling cross‐talk between RHO and RAS GTPases ...................................................... 20 Aims and objectives ................................................................................................................. 23 Chapter II ..................................................................................................................................... 24 The function of embryonic stem cell‐expressed RAS (ERAS), a unique RAS family member, correlates with its additional motifs and its structural properties ......................................... 24 Chapter III .................................................................................................................................... 38 Embryonic stem cell‐expressed ERAS controls quiescence of hepatic stellate cells............... 38 Chapter IV .................................................................................................................................... 39 Classical RHO proteins: biochemistry of molecular switch function and regulation .............. 58 Chapter V ..................................................................................................................................... 73 Functional cross‐talk between RAS and RAS pathways: a RAS‐specific GTPase activating protein (p120RASGAP) competitively inhibits the RHOGAP activity of deleted in liver cancer (DLC) tumor suppressor by masking the catalytic arginine finger ....................... 73 Chapter VI .................................................................................................................................... 85 IQGAP1 interaction with RHO family proteins revisited: kinetic and equilibrium evidence for two distinct binding sites ........................................................................................................ 85 Chapter VII ................................................................................................................................. 117 Juvenile myelomonocytic leukemia displays mutations in components of the RAS pathway and the PRC2 network ........................................................................................................... 117 Chapter VIII ................................................................................................................................ 142 General discussion ................................................................................................................. 142 Biochemical characteristic of embryonic stem cell‐expressed RAS (ERAS) .......................... 144 Signaling network and proposed function of endogenous ERAS in HSC ............................... 146 Functional cross‐talk between RAS and RHO pathways ....................................................... 150 p120GAP links RAS and RHO pathways via p190GAP and DLC1 ....................................... 151 Scaffolding protein IQGAP modulates RHO and RAS signaling ......................................... 152 The coordination of RAS (NRAS) and RHO (RAC2) mutations in tumor progression ........ 154 Concluding remarks ............................................................................................................... 155 References ................................................................................................................................. 157 Acknowledgments ..................................................................................................................... 178 Curriculum Vitae ........................................................................................................................ 180 Eidesstattliche Erklärung zur Dissertation ................................................................................ 183 Summary Hepatic stellate cells (HSCs) are non‐parenchymal liver resident cells in the space of Dissé, which are central to metabolism and storage of retinoids in the body and are involved in liver development, immunoregulation, homeostasis, regeneration, and fibrosis. In healthy liver, HSCs are in a state referred as quiescent. After liver injury, HSCs develop into activated HSCs, which are then able to proliferate, migrate, contract, and differentiate to other liver cell types and, in this way, contribute to liver regeneration. However, during sustained liver injury HSCs promote liver fibrosis via excessive extracellular matrix production. The key signaling networks, which maintain quiescent of HSCs or orchestrate their plasticity toward liver regeneration or fibrosis need further investigation. The RAS family is central in a network controlling intracellular signaling pathways, which adopt the cellular responses upon integration of external stimuli from the neighboring cells and the microenvironment. The functions and activity of RAS dependent signaling pathways in the fate of HSCs are poorly understood. This doctoral thesis provides new insights into the expression pattern, isoform specificity, activity and networking of RAS family members and their signaling components in both quiescent and activated HSCs. The obtained data revealed a differential expression pattern for RAS isoforms, where embryonic stem cell‐expressed RAS (ERAS) is specifically expressed in quiescent HSCs and becomes drastically down‐regulated after HSC activation. In contrast to ERAS, other members of the RAS family, e.g., MRAS, RRAS, RALA, and RAP2A were rather up‐regulated upon HSC activation. Comprehensive biochemical studies identified ERAS as a unique member of the RAS family with remarkable sequence deviations, additional motifs, and an extended N‐terminal region. The latter appears to be important for the signaling activity of ERAS. Most remarkably, ERAS revealed a different mode of effector interaction as compared to classical HRAS signaling, thereby, correlating with deviations in the effector‐ binding site of ERAS. Hence, ERAS signals maintain the HSC quiescent by the inhibition of both proliferation and apoptosis via various pathways, such as JAK‐STAT3, AKT‐mTORC1‐FOXO1, mTORC2‐AKT, and RASSF5‐HIPPO. In contrast, activated HSCs exhibited YAP‐CTGF/NOTCH2 and RAS‐RAF‐MEK‐ERK activity, which are involved in HSC proliferation and development. I Zusammenfassung Hepatische Sternzellen (HSCs) sind nicht‐parenchymale, im Dissé'schen Raum der Leber ansässige Zellen, welche für den Metabolismus und die Speicherung von Retinoiden im
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