@phdthesis{Ullrich2014, author = {Ullrich, Melanie}, title = {Identification of SPRED2 as a Novel Regulator of Hypothalamic-Pituitary-Adrenal Axis Activity and of Body Homeostasis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-107355}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach. An initial phenotypical characterization of KO mice aged up to five months identified SPRED2 as a regulator of chondrocyte differentiation and bone growth. Here, the loss of SPRED2 leads to an augmented FGFR-dependent ERK activity, which in turn causes hypochondroplasia-like dwarfism. However, long term observations of older KO mice revealed a generally bad state of health and manifold further symptoms, including excessive grooming associated with severe self-inflicted wounds, an abnormally high water uptake, clear morphological signs of kidney deterioration, and a reduced survival due to sudden death. Based on these observations, the aim of this study was to discover an elicitor of this complex and versatile phenotype. The observed kidney degeneration in our SPRED2 KO mice was ascribed to hydronephrosis characterized by severe kidney atrophy and apoptosis of renal tubular cells. Kidney damage prompted us to analyze drinking behavior and routine serum parameters. Despite polydipsia, which was characterized by a nearly doubled daily water uptake, the significantly elevated Na+ and Cl- levels and the resulting serum hyperosmolality could not be compensated in SPRED2 KOs. Since salt and water balance is primarily under hormonal control of aldosterone and AVP, we analyzed both hormone levels. While serum AVP was similar in WTs and KOs, even after experimental water deprivation and an extreme loss of body fluid, serum aldosterone was doubled in SPRED2 KO mice. Systematic investigation of contributing upstream hormone axes demonstrated that hyperaldosteronism developed independently of an overactivated Renin-Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. Serum corticosterone, which is like aldosterone released from the adrenal cortex, was more than doubled in SPRED2 KOs, too. Similar to corticosterone, the production of aldosterone is at least in part under control of pituitary ACTH, which is further regulated by upstream hypothalamic CRH release. In fact, stress hormone secretion from this complete hypothalamic-pituitary-adrenal axis was upregulated because serum ACTH, the mid acting pituitary hormone, and hypothalamic CRH, the upstream hormonal inductor of HPA axis activity, were also elevated by 30\% in SPRED2 KO mice. This was accompanied by an upregulated ERK activity in paraventricular nucleus-containing hypothalamic brain regions and by augmented hypothalamic CRH mRNA levels in our SPRED2 KO mice. In vitro studies using the hypothalamic cell line mHypoE-44 further demonstrated that both SPRED1 and SPRED2 were able to downregulate CRH promoter activity, CRH secretion, and Ets factor-dependent CRH transcription. This was in line with the presence of various Ets factor binding sites in the CRH promoter region, especially for Ets1. Thus, this study shows for the first time that SPRED2-dependent inhibition of Ras/ERK/MAPK signaling by suppression of ERK activity leads to a downregulation of Ets1 factor-dependent transcription, which further results in inhibition of CRH promoter activity, CRH transcription, and CRH release from the hypothalamus. The consecutive hyperactivity of the complete HPA axis in our SPRED2 KO mice reflects an elevated endogenous stress response becoming manifest by excessive grooming behavior and self-inflicted skin lesions on the one hand; on the other hand, in combination with elevated aldosterone synthase expression, this upregulated HPA hormone release explains hyperaldosteronism and the associated salt and water imbalances. Both hyperaldosteronism and polydipsia very likely contribute further to the observed kidney damage. Taken together, this study initially demonstrates that SPRED2 is essential for the appropriate regulation of HPA axis activity and of body homeostasis. To further enlighten and compare consequences of SPRED2 deficiency in mice and particularly in humans, two follow-up studies investigating SPRED2 function especially in heart and brain, and a genetic screen to identify human SPRED2 loss-of-function mutations are already in progress.}, subject = {Renin-Angiotensin-System}, language = {en} } @article{RajendranBoettigerStadelmannetal.2021, author = {Rajendran, Ranjithkumar and B{\"o}ttiger, Gregor and Stadelmann, Christine and Karnati, Srikanth and Berghoff, Martin}, title = {FGF/FGFR pathways in multiple sclerosis and in its disease models}, series = {Cells}, volume = {10}, journal = {Cells}, number = {4}, issn = {2073-4409}, doi = {10.3390/cells10040884}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236594}, year = {2021}, abstract = {Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS) affecting more than two million people worldwide. In MS, oligodendrocytes and myelin sheaths are destroyed by autoimmune-mediated inflammation, while remyelination is impaired. Recent investigations of post-mortem tissue suggest that Fibroblast growth factor (FGF) signaling may regulate inflammation and myelination in MS. FGF2 expression seems to correlate positively with macrophages/microglia and negatively with myelination; FGF1 was suggested to promote remyelination. In myelin oligodendrocyte glycoprotein (MOG)\(_{35-55}\)-induced experimental autoimmune encephalomyelitis (EAE), systemic deletion of FGF2 suggested that FGF2 may promote remyelination. Specific deletion of FGF receptors (FGFRs) in oligodendrocytes in this EAE model resulted in a decrease of lymphocyte and macrophage/microglia infiltration as well as myelin and axon degeneration. These effects were mediated by ERK/Akt phosphorylation, a brain-derived neurotrophic factor, and downregulation of inhibitors of remyelination. In the first part of this review, the most important pharmacotherapeutic principles for MS will be illustrated, and then we will review recent advances made on FGF signaling in MS. Thus, we will suggest application of FGFR inhibitors, which are currently used in Phase II and III cancer trials, as a therapeutic option to reduce inflammation and induce remyelination in EAE and eventually MS.}, language = {en} } @article{RadevaWalterStachetal.2019, author = {Radeva, Mariya Y. and Walter, Elias and Stach, Ramona Alexandra and Yazdi, Amir S. and Schlegel, Nicolas and Sarig, Ofer and Sprecher, Eli and Waschke, Jens}, title = {ST18 Enhances PV-IgG-Induced Loss of Keratinocyte Cohesion in Parallel to Increased ERK Activation}, series = {Frontiers in Immunology}, volume = {10}, journal = {Frontiers in Immunology}, doi = {10.3389/fimmu.2019.00770}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224910}, pages = {770, 1-11}, year = {2019}, abstract = {Pemphigus is an autoimmune blistering disease targeting the desmosomal proteins desmoglein (Dsg) 1 and Dsg3. Recently, a genetic variant of the Suppression of tumorigenicity 18 (ST18) promoter was reported to cause ST18 up-regulation, associated with pemphigus vulgaris (PV)-IgG-mediated increase in cytokine secretion and more prominent loss of keratinocyte cohesion. Here we tested the effects of PV-IgG and the pathogenic pemphigus mouse anti-Dsg3 antibody AK23 on cytokine secretion and ERK activity in human keratinocytes dependent on ST18 expression. Without ST18 overexpression, both PV-IgG and AK23 induced loss of keratinocyte cohesion which was accompanied by prominent fragmentation of Dsg3 immunostaining along cell borders. In contrast, release of pro-inflammatory cytokines such as IL-1 alpha, IL-6, TNF alpha, and IFN-gamma was not altered significantly in both HaCaT and primary NHEK cells. These experiments indicate that cytokine expression is not strictly required for loss of keratinocyte cohesion. Upon ST18 overexpression, fragmentation of cell monolayers increased significantly in response to autoantibody incubation. Furthermore, production of IL-1 alpha and IL-6 was enhanced in some experiments but not in others whereas release of TNF-alpha dropped significantly upon PV-IgG application in both EV- and ST18-transfected HaCaT cells. Additionally, in NHEK, application of PV-IgG but not of AK23 significantly increased ERK activity. In contrast, ST18 overexpression in HaCaT cells augmented ERK activation in response to both c-IgG and AK23 but not PV-IgG. Because inhibition of ERK by U0126 abolished PV-IgG- and AK23-induced loss of cell cohesion in ST18-expressing cells, we conclude that autoantibody-induced ERK activation was relevant in this scenario. In summary, similar to the situation in PV patients carrying ST18 polymorphism, overexpression of ST18 enhanced keratinocyte susceptibility to autoantibody-induced loss of cell adhesion, which may be caused in part by enhanced ERK signaling.}, language = {en} } @phdthesis{Nekhoroshkova2009, author = {Nekhoroshkova, Elena}, title = {A-RAF kinase functions in ARF6 regulated endocytic membrane traffic}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-44566}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Extracellular signals are translated and amplified via cascades of serially switched protein kinases, MAP kinases (MAPKs). One of the MAP pathways, the classical RAS/RAF/MEK/ERK pathway, transduces signals from receptor tyrosine kinases and plays a central role in regulation of cell proliferation. RAF kinases (A-, B- and C-RAF) function atop of this cascade and convert signals emanating from conformational change of RAS GTPases into their kinase activity, which in turn phosphorylates their immediate substrate, MEK. Disregulated kinase activity of RAF can result in tumor formation, as documented for many types of cancer, predominantly melanomas and thyroid carcinomas (B-RAF). A-RAF is the least characterized RAF, possibly due to its low intrinsic kinase activity and comparatively mild phenotype of A-RAF knockout mice. Nevertheless, the unique phenotype of araf -/- mice, showed predominantly neurological abnormalities such as cerebellum disorders, suggesting that A-RAF participates in a specific process not complemented by activities of B- and CRAF. Here we describe the role of A-RAF in membrane trafficking and identify its function in a specific step of endocytosis. This work led to the discovery of a C-terminally truncated version of A-RAF, AR149 that strongly interfered with cell growth and polarization in yeast and with endocytosis and actin polymerization in mammalian cells. As this work was in progress two splicing isoforms of ARAF, termed DA-RAF1 and DA-RAF2 were described that act as natural inhibitors of RAS-ERK signaling during myogenic differentiation (Yokoyama et al., 2007). DA-RAF2 contains the first 153 aa of A-RAF and thus is nearly identical with AR149. AR149 localized specifically to the recycling endosomal compartments as confirmed by colocalization and coimmunoprecipitation with ARF6. Expression of AR149 interferes with recycling of endocytosed transferrin (Tfn) and with actin polymerization. The endocytic compartment, where internalized Tfn is trapped, was identified as ARF6- and RAB11- positive endocytic vesicles. We conclude that the inhibition of Tfn trafficking in the absence of A-RAF or under overexpression of AR149 occurs between tubular- and TGNassociated recycling endosomal compartments. siRNA-mediated depletion of endogenous A-RAF or inhibition of MEK by U0126 mimic the AR149 overexpression phenotype, supporting a role of ARAF regulated ERK signalling at endosomes that is controlled by AR149 and targets ARF6. Our data additionally suggest EFA6 as a partner of A-RAF during activation of ARF6. The novel findings on the A-RAF localization and the interaction with ARF6 have led to a new model of ARAF function were A-RAF via activation of ARF6 controls the recycling of endocytic vesicles.Endocytosis and rapid recycling of synaptic vesicles is critically important for the physiological function of neurons. The finding, that A-RAF regulates endocytic recycling open a new perspective for investigation of the role of A-RAF in the nervous system.}, subject = {Raf-Kinasen}, language = {en} }