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The prototyical tumor suppressor p53 is able to arrest cells after DNA damage or as a response to oncogene expression. The transactivation-competent (TA) isoforms of the more recently discovered p53 family member p73 also prevent tumors, but the underlying mechanisms are less well understood. The work presented here addressed this issue by using a cell culture model of tumorigenesis in which normal human diploid fibroblasts are stepwise transduced with oncogenes. Cells in pretransformed stages were shown to harbour high levels of TAp73 mRNA and protein. This positive regulation was probably a result of pRB inactivation and derepression of E2F1, a key activator of TAp73. Consequences for such cells included an increased sensitivity to the cytostatic drug adriamycin, slower proliferation and reduced survival at high cell density, as demonstrated by rescue experiments using siRNA-mediated knockdown of TAp73. In order to identify potential effector pathways, the gene expression profile of siRNA treated, matched fibroblast cell lines with high and low TAp73 levels were compared in DNA microarrays. These findings support the notion of TAp73 up-regulation as an anti-proliferative defense mechanism, blocking the progress towards full transformation. This barrier could be overcome by the introduction of a constitutively active form of Ras which caused a switch from TAp73 to oncogenic DeltaNp73 expression, presumably through the phosphatidylinositol 3-kinase (PI3K) pathway. In summary, the results presented emphasize the tumor-suppressive function of TAp73 and indicate that its downregulation is a decisive event during the transformation of human cells by oncogenic Ras mutants.
The Transforming Growth Factor (TGF) superfamily of cytokines and their serine/threonine kinase receptors play an important role in the regulation of cell division, differentiation, adhesion, migration, organization, and death. Smad proteins are the major intracellular signal transducers for the TGF receptor superfamily that mediate the signal from the membrane into the nucleus. Bone Morphogenetic Protein-4 (BMP-4) is a representative of the TGF superfamily, which regulates the formation of teeth, limbs and bone, and also plays a role in fracture repair. Binding of BMP-4 to its receptor stimulates phosphorylation of Smad1, which subsequently recruits Smad4. A hetero-oligomeric complex consisting of Smad1 and Smad4 then translocates into the nucleus and regulates transcription of target genes by interacting with transcription factors. Although the individual steps of the signaling cascade from the receptor to the nucleus have been identified, the exact kinetics and the rate limiting step(s) have remained elusive. Standard biochemical techniques are not suitable for resolving these issues, as they do not offer sufficiently high sensitivity and temporal resolution. In this study, advanced optical techniques were used for direct visualization of Smad signaling in live mammalian cells. Novel fluorescent biosensors were developed by fusing cyan and yellow fluorescent proteins to the signaling molecules Smad1 and Smad4. By measuring Fluorescence Resonance Energy Transfer (FRET) between the two fluorescent proteins, the kinetics of BMP/Smad signaling was unraveled. A rate-limiting delay of 2 - 5 minutes occurred between BMP receptor stimulation and Smad1 activation. A similar delay was observed in the complex formation between Smad1 and Smad4. Further experimentation indicated that the delay is dependent on the Mad homology 1 (MH1) domain of Smad1. These results give new insights into the dynamics of the BMP receptor – Smad1/4 signaling process and provide a new tool for studying Smads and for testing inhibitory drugs.
Platelets are crucial to inhibit extensive blood loss at sites of vascular injury. However, under pathological conditions such as rupture of an atherosclerotic plaque, activated platelets form aggregates that may occlude the vessel. This can lead to heart attack and stroke. Various and complex signaling pathways in the cell are involved in the steps of platelet adhesion, activation and aggregation. Single aspects of these processes were studied in three different subprojects in this work. The Glycoprotein (GP) Ib-V-IX complex is responsible for the first contact of platelets with the vessel wall. Subsequently, GPVI can bind to collagen of the subendothelium, which initiates a signaling cascade leading to platelet activation, aggregation, characterized by integrin activation and granule secretion and platelet procoagulant activity. The latter is characterized by exposed phosphatidylserine (PS) on the platelet surface, which enhances thrombin generation and thereby the coagulation cascade. A controlled regulation of GP receptors on the platelet surface is vital for an intact response of the cell to platelet agonists. In the first subproject described here the regulation of GPV and GPVI on mouse platelets was investigated and it was found that both receptors are shed from the platelet surface in a metalloproteinase dependent manner. However, GPVI is shed upon mitochondrial injury, while GPV cleavage could be observed upon platelet stimulation. The metalloproteinase responsible for GPVI shedding remains unknown whereas the metallproteinase that sheds GPV was identified in this work as being ADAM17. This shows that the expression of both receptors underlies a controlled mechanism regulated through distinct metalloproteinases. In the second subproject the role of protein kinase C (PKC) in platelet activation and procoagulant response was investigated using PKC specific inhibitors. It was found that PKC blockage reduced platelet activation but enhanced platelet procoagulant activity. This is the first time that a dual role in platelet activation and procoagulant activity is defined for PKC. In the third project the role of the small GTPase Rac1 in platelet signaling was studied using conditional Rac1 knock out mice. It is reported here that Rac1 lies downstream of GPVI and is involved in integrin activation and cytsolic Ca2+ changes in vitro and platelet adhesion and thrombus formation in vivo. This is the first time that Rac1 is demonstrated to have a pivotal role in GPVI signaling and furthermore points to a novel, unknown pathway downstream of GPVI.
Krebserkrankungen zeichnen sich häufig durch Störungen zellulärer Differenzierungsprozesse aus. So weisen Rhabdomyosarkome, die aus Muskelvorläuferzellen hervorgehen, Differenzierungsdefekte auf, die zur unkontrollierten Proliferation der Tumorzellen führen. Bislang ist ungeklärt, ob die Differenzierungsdefekte auf der verstärkten Expression von Inhibitoren, der defekten Funktion von Aktivatoren oder einer Kombination von beidem beruht. In dieser Arbeit wird gezeigt, dass im Unterschied zu normalen Muskelzellen RMS-Zellen verstärkt DeltaNp73, einen Pan-Inhibitor der p53-Tumorsuppressorfamilie, exprimieren. Die experimentelle Überexpression von DeltaNp73 in normalen Myoblasten blockierte die Muskeldifferenzierung und förderte in Kombination mit klassischen RMS-Onkogenen wie IGF2 oder PAX3/FKHR die maligne Transformation. Umgekehrt führte die Hemmung von DeltaNp73 durch RNAi zur Reduktion der Tumorigenität von RMS-Tumorzellen. Da DeltaNp73 als dominant-negativer Inhibitor der p53-Familie wirkt, lies die Hemmung von Differenzierungsprozessen durch DeltaNp73 vermuten, dass die p53-Familienmitglieder (p53, p63, und p73) an der Regulation der Muskeldifferenzierung beteiligt sind. Tatsächlich konnte in dieser Arbeit gezeigt werden, dass die drei p53-Familienmitglieder bei der Induktion später Differenzierungsstadien kooperieren, indem sie die Aktivität des Retinoblastoma-Proteins RB regulieren. Die Funktion von RB ist bekanntermassen sowohl für den permanenten Zellzyklusarrest als auch für die Aktivierung Muskel-spezifischer Gene notwendig. Während p53 die Proteinspiegel von RB reguliert, kontrollieren p63 und p73 den Aktivierungsgrad von RB, indem sie dessen Phoshphorylierungszustand über den Zyklin-abhängigen Kinaseinhibitor p57KIP2 modifizieren. Eine Hemmung dieser Funktionen blockiert das Differenzierungsprogramm und fördert die Tumorentstehung. Die Aktivierung zellulärer Differenzierungsprozesse stellt somit einen entscheidenden Bestandteil der Tumorsuppressoraktivität der p53-Familie dar und liefert eine Erklärung für die Häufigkeit von Mutationen im p53-Signalweg bei Rhabdomyosarkom-Patienten.
Human cytomegalovirus (HCMV) infection causes clinical symptoms in immunocompromised individuals such as transplantant recipients and AIDS patients. The virus is also responsible for severe complications in unborn children and young infants. The species specificity of HCMV prevents the direct study of mechanisms controlling the infection in animal models. Instead, the murine cytomegalovirus (MCMV) is used as a model system. Human and murine CMVs have large double-stranded DNA genomes, encoding nearly 170 genes. About 30% of the genes are committed to essential tasks of the virus. The remaining genes are involved in virus pathogenesis or host interaction and are dispensable for virus replication. The CMV genes are classified in gene families, based on sequence homology. In the present work, the function of two genes of the US22 gene family was analyzed. The MCMV genes m142 and m143 are the only members of this family that are essential for virus replication. These genes also differ from the remaining ten US22 gene family members in that they lack 1 of 4 conserved sequence motifs that are characteristic of this family. The same conserved motif is missing in the HCMV US22 family members TRS1 and IRS1, suggesting a possible functional homology. To demonstrate an essential role of m142 and m143, the genes were deleted from the MCMV genome, and the mutants were reconstituted on complementing cells. Infection of non-complementing cells with the deletion mutants did not result in virus replication. Virus growth was rescued by reinsertion of the corresponding genes. Cells infected with the viral deletion mutants synthesized reduced amounts of viral DNA, and viral late genes were not expressed. However, RNA analyses showed that late transcripts were present, excluding a role of m142 and m143 in regulation of gene transcription. Metabolic labelling experiments showed that total protein synthesis at late times postinfection was impaired in cells infected with deletion mutants. Moreover, the dsRNA-dependent protein kinase R (PKR) and its target protein, the translation initiation factor 2α (eIF2α) were phosphorylated in these cells. This suggested that the m142 and m143 are required for blocking the PKR-mediated shut-down of protein synthesis. Expression of the HCMV gene TRS1, a known inhibitor of PKR activation, rescued the replication of the deletion mutants, supporting the observation that m142 and m143 are required to inhibit this innate immune response of the host cell.
Viren durchliefen eine gemeinsame Evolution mit ihren Wirtsorganismen, die zu einer spezifischen Anpassung der Viren an ihren jeweiligen Wirt führte. Als Folge dessen verfügen viele Viren über ein eng begrenztes Wirtsspektrum. Gelegentlich machen Viren Veränderungen durch, die es ihnen erlauben, einen neuen Wirt zu infizieren und in ihm zu replizieren, wie dies in jüngster Vergangenheit beim humanen Immundefizienz-Virus oder beim Grippevirus geschehen ist. Spezies-übergreifende Infektionen sind für die meisten neuen und wiederauftauchenden Viruserkrankungen verantwortlich. Allerdings ist bisher wenig über die Mechanismen bekannt, die Viren auf einen bestimmten Wirt beschränken, und welche Faktoren Viren zur Überwindung der Spezies-Barriere und zur Vermehrung in einer neuen Wirtsspezies benötigen. Cytomegaloviren sind Prototypen der beta-Herpesvirus Unterfamilie und verfügen über eine ausgeprägte Spezies-Spezifität. Sie vermehren sich nur in Zellen der eigenen oder einer eng verwandten Wirtsspezies. Der molekulare Mechanismus, der dieser Spezies-Spezifität zugrunde liegt, ist noch weitgehend unbekannt und stellt deshalb das Thema dieser Arbeit dar. Initiale Beobachtungen zeigten, dass sich das Maus-Cytomegalovirus (MCMV) ausschließlich in menschlichen 293 und 911 Zellen, aber keiner anderen getesteten menschlichen Zelle vermehren ließ. Diese beiden Zelllinien sind mit Adenovirus E1-Genen transformiert, die den Transkriptions-Transaktivator E1A sowie zwei Apoptose-Inhibitoren (E1B-55k und E1B-19k) kodieren. Daher lag die Hypothese nahe, dass diese Funktionen benötigt werden, um eine MCMV-Replikation in menschlichen Zellen zu ermöglichen. Außerdem konnte gezeigt werden, dass normale menschliche Zellen nach Infektion rapide absterben, und zwar durch eine Caspase-9-vermittelte Apoptose. Die Induktion der Apoptose durch MCMV lässt sich durch Caspase-Inhibitoren unterdrücken, wodurch die virale Replikation wiederhergestellt wird. Dies deutet auf eine Schlüsselfunktion der Caspasen für diesen Prozess hin. Durch Überexpression eines mitochondrialen Apoptose-Inhibitors, d.h. eines Bcl-2-ähnlichen Proteins, in menschlichen Zellen ließ sich die Virus-induzierte Apoptose verhindern. Diese Zellen erlaubten ebenfalls eine effiziente MCMV-Replikation. Die Bedeutung Bcl-2-ähnlicher Proteine für die Spezies-übergreifende Cytomegalovirus-Infektion wurde sowohl durch die Integration korrespondierender Gene, alsauch durch die Integration anderer Inhibitioren der Apoptose oder von Kontroll-Genen in das MCMV Genom bestätigt. Nur rekombinante Viren, die ein Bcl-2-ähnliches Protein kodieren, konnten in menschlichen Zellen vermehrt werden. Ein einziges Gen des humanen Cytomegalovirus, das einen mitochondrialen Apoptose-Inhibitor kodiert, reichte aus, um eine MCMV-Replikation in menschlichen Zellen zu ermöglichen. Zusätzlich konnte gezeigt werden, dass dieselben Prinzipien für eine Replikation des Ratten-Cytomegalovirus in menschlichen Zellen gelten. Zusammenfassend kann festgestellt werden, dass die Induktion der Apoptose eine Spezies-übergreifende Infektion bei den Nagetier-Cytomegaloviren einschränkt.
Das Ribonukleoprotein, Telomerase wird vor allem für die Aufrechterhaltung der Telomerlänge benötigt und ist normalerweise nur in Keimbahnzellen, Stammzellen und anderen Zellen mit erhöhter Regenerationsfähigkeit aktiv. Die Aktivierung der Telomerase ist darüber hinaus ein wichtiger Faktor während der Krebsentstehung. Fast das komplette Spektrum humaner Tumore zeichnet sich durch hohe Telomerase-Aktivität aus. Vor allem maligne Tumore besitzen eine sehr aktive Telomerase, unlimitiertes Wachstum und Immortalität ermöglicht. Die Aktivität der Telomerase wird vor allem über die Expression der katalytischen Untereinheit hTERT reguliert, die unter der strikten Kontrolle verschiedener Tumorsuppressorgene liegt. Zu den wichtigsten Regulatoren der hTERT-Expression gehört auch der bekannte Tumorsuppressor p53. Über die Rolle des p53-Familienmitglieds p73 in der Regulation der Telomerase-Aktivität war bisher nur wenig bekannt. Im Rahmen dieser Arbeit konnte ein regulatorischer Einfluss von p73 nachgewiesen werden. Dabei wurden deutliche Unterschiede in der Funktion der N-terminalen Isoformen TAp73 und DeltaNp73 beobachtet. TAp73 erwies sich sowohl nach Überexpression als auch nach Induktion des endogenen TAp73 als ein effizienter Repressor der hTERT-Expression. Im Gegensatz dazu konnte durch die Hemmung des endogenen TAp73 mittels RNAi die Expression von hTERT in verschiedenen Zelllinen induziert werden. Zusätzlich zu der Funktion als Tumorsuppressor scheint p73 auch in verschiedene Differenzierungsprozesse involviert zu sein. Die Expression von p73 korreliert zwar mit der Hemmung der Telomerase-Aktivität während der myeloischen Differenzierung von HL60-Zellen, hat hier aber keine Bedeutung für die Repression von hTERT. Die N-terminal verkürzte Isoform DeltaNp73 wirkt im Gegensatz zu TAp73 als effizienter Aktivator der hTERT-Expression. DeltaNp73 induziert die hTERT-Expression einerseits über seine dominant-negative Funktion auf die pro-apoptotischen p53-Familienmitglieder und andererseits über die Hemmung repressiver RB-E2F-Komplexe. Im Rahmen dieser Studie erwies sich p73 somit als ein wichtiger Regulator der Telomerase Aktivität, wobei sich eine duale Rolle als negativer (TAp73) und auch als positiver (DeltaNp73) Regulator der Telomerase Aktivität herausstellte.
Platelet activation and adhesion resulting in thrombus growth is essential for normal hemostasis, but can lead to irreversible, life-threatening vessel occlusion. In the current study, the contribution of platelet integrins, activation receptors and the contact system of blood coagulation in such pathological conditions was investigated in mice.
Platelet interaction with the subendothelium is essential to limit blood loss after tissue injury. However, upon rupture of atherosclerotic plaques, this interaction may result in blood vessel occlusion leading to life threatening diseases such as myocardial infarction or stroke. Among the subendothelial matrix proteins, collagen is considered to be the most thrombogenic component as it directly activates platelets. Platelets interact with collagen, either indirectly through glycoprotein (GP) Ib-V-IX receptor complex, or directly through the major collagen receptor on the platelet surface, GPVI. The work presented here focused on studying the cellular regulation of GPVI. In addition, a possible role for GPVI in thrombus formation induced by atherosclerotic plaque material was investigated and it was found that GPVI plays an important role in this process. Using a recently published mitochondrial injury model, it was found that GPVI contains a cleavage site for a platelet-expressed metalloproteinase. Further studies showed that platelet activation by CRP, or thrombin induced down-regulation of GPIb, but not GPVI. In parallel, cellular regulation of GPV was studied and it was found that GPV is cleaved in vitro by the metalloproteinase ADAM17. In previous studies it was shown that injection of mice with the anti-GPVI mAb, JAQ1, induces GPVI down-regulation, which is associated with a strong, but transient, thrombocytopenia. Using new anti-GPVI mAbs, which bind different epitopes on the receptor, it is shown in this study that GPVI down-regulation occurs in an epitope-independent manner. Further experiments showed that antibody treatment induces a transient, but significant increase in bleeding time. Using different genetically modified mice, it is shown that, upon antibody injection, GPVI is both, shed from the platelet surface and internalized into the platelet. Signaling through the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR chain is essential for both processes, while LAT and PLC2 are essential for the shedding process only. Antibody-induced increase in bleeding time and thrombocytopenia were absent in LAT deficient mice, showing that it is possible to uncouple the associated side effects from the down-regulation process. As antibody-induced GPVI internalization still occurs in LAT and PLC2 deficient mice, this suggests a novel signaling pathway downstream of GPVI that has not been described so far.
In vitro and in vivo studies on the activating platelet collagen receptor glycoprotein VI in mice
(2003)
The work summarized here focused on the characterization of the murine platelet collagen receptor glycoprotein (GP) VI and was performed to evaluate its potential as an antithrombotic target. The first mAb against (mouse) GPVI, JAQ1, was generated and used to demonstrate that GPVI requires the FcRgamma-chain for its expression and function and that this receptor is the central molecule in collagen-induced platelet activation. Blocking the major collagen binding site on GPVI with JAQ1 revealed the presence of a second activatory epitope within collagen. Additionally, the collagen receptor integrin alpha2beta1 was found to be required for activation via this second pathway but not to be essential for collagen-induced activation of normal platelets. In studies with mice expressing reduced levels of the GPVI-FcRgamma-complex, differential responses to GPVI ligands were observed. Most importantly, the striking difference between platelet responses to collagen and the GPVI specific synthetic collagen related peptide (CRP) confirmed the supportive role of other collagen receptor(s) on platelets. Irrespective of yet undefined additional receptors, studies with mice deficient in GPVI (FcRgamma-chain) or alpha2beta1 showed that GPVI, but not alpha2beta1 is essential for platelet-collagen interaction. Based on these results, the model of platelet attachment to collagen was revised establishing GPVI as the initial activating receptor which upregulates the activity of integrins, thus enabling firm attachment of platelets to the ECM. While the mAb JAQ1 had only limited inhibitory effects on collagen-induced activation in vitro, its in vivo application to mice resulted in completely abolished platelet responses to collagen and the GPVI specific agonists CRP and convulxin. This effect was found to be due to antibody-induced irreversible down-regulation of GPVI on circulating platelets for at least two weeks. Further studies revealed that GPVI depletion occurs independently of the targeted epitope on the receptor and does not require the divalent form of IgG as it was also induced by mAbs (JAQ2, JAQ3) or the respective Fab fragments directed against epitopes distinct from the major collagen binding site. The internalization of GPVI in vivo resulted in a long-term protection of the mice from lethal collagen-dependent thromboembolism whereas it had only moderate effects on the bleeding time, probably because the treatment did not affect other activation pathways. These results establish GPVI as a potential pharmacological target for the prevention of ischemic cardiovascular diseases and may open the way for a completely new generation of antithrombotics.