Die Rolle von CXCR4 und Gab1 bei der Entwicklung von Muskelvorläuferzellen

1\. Title and Summary 1 2\. Introduction 5 3\. Materials and Methods 17 4\. Results 44 5\. Discussion 67 6\. References 80 Acknowledgments, Declaration 88By the use of the microarray technology I have determined the gene expression profile of migrating muscle precursors of Lbx1GFP/+ and Lbx1GFP/GFP mutant mice. Among the expressed genes, the chemokine receptor CXCR4 was identified. Expression of CXCR4 was downregulated in Lbx1GFP/GFP mutant mice in muscle precursors of the limb, but not in those cells that migrate to the tongue anlage. The CXCR4 ligand, SDF1, is expressed in the mesenchyme of the limb and the first branchial arch, the targets of the migrating cells. In my thesis, I analyzed the function of CXCR4 signaling in the development of migrating muscle precursors. Gain-of-function experiments of chick embryos demonstrate that SDF1 can provide attractive cues for muscle precursor cells and suppress their differentiation. Analysis of CXCR4 mutant mice demonstrates changes in the distribution of migrating muscle precursors in the dorsal limb and branchial arches. These changes were accompanied by increased apoptosis, indicating that CXCR4 signals provide not only attractive cues but control also survival. Furthermore, I found that CXCR4 and Gab1 interact genetically. Both CXCR4 and Gab1 mutations interfere with appropriate distribution and survival of migrating muscle precursor cells. However, in CXCR4;Gab1 mutant mice the migrating muscle precursors are affected more strongly than in either of the single mutants. For instance, migrating muscle precursor cells arrive at the anlage of the tongue in CXCR4 and Gab1 mutant mice, but fail to reach this site in the CXCR4;Gab1 double mutant mice. The genetic interaction of CXCR4 and Gab1 might reflect a cross-talk between signaling cascades employed by G-protein coupled receptors and tyrosine kinases.Das Homeobox Gen Lbx1wird ausschliesslich in wandernden Muskelvorläuferzellen, nicht aber in anderen Muskelzellen exprimiert. In meiner Doktorarbeit habe einen Mausstamm, der ein Lbx1GFP Allel trägt, konstruiert und diesen Tiere benutzt, um GFP+ Muskelvorläuferzellen durch FACS sorting zu isolieren. Mittels DNA-Microarry-Technologie erstellte dann ich das Genexpressionsprofil von wandernden Muskelvorläuferzellen aus Lbx1GFP/+ und Lbx1GFP/GFP Embryonen. So konnte ich bestimmen, dass der Chemokine-Rezeptor CXCR4 in Muskelvorläuferzellen von Lbx1GFP/GFP mutanten Mäusen schwächer exprimiert ist als in den Zellen der Lbx1GFP/+ Kontrolltiere. SDF1, der Ligand von CXCR4 in den Zielgebieten der wandernden Muskelvorläuferzellen, dem Mesenchym der Extremitäten und Branchialbögen exprimiert. Gain-of-function Experimente an Hühnerembryonen zeigen, dass SDF1 als chemoattraktives Signal für Muskelvorläuferzellen wirkt und eine Differenzierung der Zellen verhindert. In CXCR4 mutanten Mäusen ist die Verteilung der Muskelvorläuferzellen in den dorsalen Extremitäten und den Branchialbögen fehlerhaft. Diese Änderung geht mit einer verstärkten Apoptoserate einher. Dies zeigt, dass CXCR4 sowohl für die Wanderung, als auch für das Überleben der Muskelvorläuferzellen eine Rolle spielt. Zudem konnte ich in meiner Arbeit eine genetische Interaktion zwischen CXCR4 und Gab1 nachweisen. Sowohl die CXCR4 als auch die Gab1 Mutation führt zu einem veränderten Verteilung und einer erhöhten Apopotose der Muskelvorläuferzellen. Allerdings ist in CXCR4;Gab1 doppelmutanten Mäusen die Wanderung der Muskelvorläufer stärker beeinträchtigt als in den jeweiligen Einzelmutanten. Zum Beispiel wandern Muskelvorläufer in CXCR4;Gab1 doppeltmutanten Mäusen nicht in die Zungenanlage ein, während dies für Muskelvorläuferzellen in CXCR4 bzw. Gab1 mutanten Mäusen der Fall ist. Diese genetische Interaktion zwischen CXCR4 und Gab1 deutet auf eine Interaktion der Signalkaskaden von G-Protein gekoppelten Rezeptoren und Tyrosinkinaserezeptoren hin

Molecular mechanisms in renal degenerative disease

Chronic kidney disease (CKD) has become a major public health problem worldwide. Therefore, a considerable effort is currently directed to understand the molecular mechanisms of renal degenerative processes. Regardless of their initiating cause, all chronic kidney diseases (CKD) develop at some level organ fibrosis that interferes with kidney function. This is also true for the two most common inherited CKD syndromes, nephronophthitis and polycystic kidney disease, whose primary defects reside within the cilium of kidney epithelial cells. A cohort of elegant recent studies has elicited the role of the primary cilium as a versatile mechanosensory organelle that also might coordinate cross-talk between multiple signaling pathways. In addition, epigenetic mechanisms are now realized to be essential in the maintenance of adult renal architecture. In this review, we will discuss recent advances in our understanding of the signaling systems implicated in kidney homeostasis and repair. (C) 2010 Elsevier Ltd. All rights reserved

CXCR4 and Gab1 cooperate to control the development of migrating muscle progenitor cells

Long-range migrating progenitor cells generate hypaxial muscle, for instance the muscle of the limbs, hypoglossal cord, and diaphragm. We show here that migrating muscle progenitors express the chemokine receptor CXCR4. The corresponding ligand, SDF1, is expressed in limb and branchial arch mesenchyme; i.e., along the routes and at the targets of the migratory cells. Ectopic application of SDF1 in the chick limb attracts muscle progenitor cells. In CXCR4 mutant mice, the number of muscle progenitors that colonize the anlage of the tongue and the dorsal limb was reduced. Changes in the distribution of the muscle progenitor cells were accompanied by increased apoptosis, indicating that CXCR4 signals provide not only attractive cues but also control survival. Gab1 encodes an adaptor protein that transduces signals elicited by tyrosine kinase receptors, for instance the c-Met receptor, and plays a role in the migration of muscle progenitor cells. We found that CXCR4 and Gab1 interact genetically. For instance, muscle progenitors do not reach the anlage of the tongue in CXCR4;Gab1 double mutants; this target is colonized in either of the single mutants. Our analysis reveals a role of SDF1/CXCR4 signaling in the development of migrating muscle progenitors and shows that a threshold number of progenitor cells is required to generate muscle of appropriate size

Nuclear movement during myotube formation is microtubule and dynein dependent and is regulated by Cdc42, Par6 and Par3

Cells actively position their nucleus within the cytoplasm. One striking example is observed during skeletal myogenesis. Differentiated myoblasts fuse to form a multinucleated myotube with nuclei positioned in the centre of the syncytium by an unknown mechanism. Here, we describe that the nucleus of a myoblast moves rapidly after fusion towards the central myotube nuclei. This movement is driven by microtubules and dynein/dynactin complex, and requires Cdc42, Par6 and Par3. We found that Par6β and dynactin accumulate at the nuclear envelope of differentiated myoblasts and myotubes, and this accumulation is dependent on Par6 and Par3 proteins but not on microtubules. These results suggest a mechanism where nuclear movement after fusion is driven by microtubules that emanate from one nucleus that are pulled by dynein/dynactin complex anchored to the nuclear envelope of another nucleus

A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells

The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi) genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor-or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targetingDAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. (C) 2016 AACR

Organometallic nucleosides induce non-classical leukemic cell death that is mitochondrial-ROS dependent and facilitated by TCL1-oncogene burden

Background: Redox stress is a hallmark of the rewired metabolic phenotype of cancer. The underlying dysregulation of reactive oxygen species (ROS) is interconnected with abnormal mitochondrial biogenesis and function. In chronic lymphocytic leukemia (CLL), elevated ROS are implicated in clonal outgrowth and drug resistance. The pro-survival oncogene T-cell leukemia 1 (TCL1) is causally linked to the high threshold towards classical apoptosis in CLL. We investigated how aberrant redox characteristics and bioenergetics of CLL are impacted by TCL1 and if this is therapeutically exploitable. Methods: Bio-organometallic chemistry provided compounds containing a cytosine nucleobase, a metal core (ferrocene, ruthenocene, Fe(CO) 3), and a 5'-CH2O-TDS substituent. Four of these metal-containing nucleoside analogues (MCNA) were tested for their efficacy and mode of action in CLL patient samples, gene-targeted cell lines, and murine TCL1-transgenic splenocytes. Results: The MCNA showed a marked and selective cytotoxicity towards CLL cells. MCNA activity was equally observed in high-risk disease groups, including those of del11q/del17p cytogenetics and of clinical fludarabine resistance. They overcame protective stromal cell interactions. MCNA-evoked PARP-mediated cell death was non-autophagic and non-necrotic as well as caspase- and P53-independent. This unconventional apoptosis involved early increases of ROS, which proved indispensible based on mitigation of MCNA-triggered death by various scavengers. MCNA exposure reduced mitochondrial respiration (oxygen consumption rate; OCR) and induced a rapid membrane depolarization (Delta psi M). These characteristics distinguished the MCNA from the alkylator bendamustine and from fludarabine. Higher cellular ROS and increased MCNA sensitivity were linked to TCL1 expression. The presence of TCL1 promoted a mitochondrial release of in part caspase-independent apoptotic factors (AIF, Smac, Cytochrome-c) in response to MCNA. Although basal mitochondrial respiration (OCR) and maximal respiratory capacity were not affected by TCL1 overexpression, it mediated a reduced aerobic glycolysis (lactate production) and a higher fraction of oxygen consumption coupled to ATP-synthesis. Conclusions: Redox-active substances such as organometallic nucleosides can confer specific cytotoxicity to ROS-stressed cancer cells. Their P53- and caspase-independent induction of non-classical apoptosis implicates that redox-based strategies can overcome resistance to conventional apoptotic triggers. The high TCL1-oncogenic burden of aggressive CLL cells instructs their particular dependence on mitochondrial energetic flux and renders them more susceptible towards agents interfering in mitochondrial homeostasis