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Auf dem Weg vom Primärtumor zur systemischen Metastasierung, der Haupttodesursache von Krebserkrankungen, ist die Einzelzellmigration von Tumorzellen durch dreidimensionales Bindegewebe ein entscheidender Schritt. Die vorliegende Arbeit zeigt Untersuchungen zur Tumorzellmigration und –plastizität in einem 3D-Migrationsmodell. Kleine G-Proteine kontrollieren Zytoskelettfunktionen, insbesondere Aktinpolymerisation und die Bildung von Zellprotrusionen durch Rac sowie Actomyosinkontraktion durch Rho. Durch pharmakologische Inhibitoren von Rac und dem Rho-Effektor ROCK soll deren Bedeutung für Einzelzellmigration in einem dreidimensionalen Modell und vor allem der Effekt auf Morphologie, Plastizität und Migration von Tumorzellen geklärt werden. Nach Inhibition von ROCK zeigen hochinvasive HT1080 Fibrosarkomzellen einen multipolar-dendritischen und sessilen Phänotyp. Nach Hemmung von Rac wird hingegen ein rundlicher, aber ebenfalls apolarer und sessiler Phänotyp induziert. Bei simultaner Inhibition von Rac und ROCK entstehen rundliche, apolare, sessile Zellen mit abortiven Pseudopodien. Wird das Gleichgewicht von Rac und ROCK durch konstitutive Aktivierung von ROCK gestört, so entsteht eine zweigeteilte Population, bestehend aus rundlichen Zellen, die Blebs bilden, und langgezogenen Zellen. Nach Sortierung nach ihrem ß1-Integrinexpressionsniveau zeigten Zellen mit niedriger Integrin-Expression einen rundlichen Migrationstyp mit blasenartigen dynamischen Protrusionen, während Zellen mit hoher Integrin-Expression langgezogen-mesenchymal migrierten. Somit steuern ROCK und Rac gemeinsam und zeitgleich die mesenchymale Einzelzellmigration. Während Rac Protrusion vermittelt, ist ROCK für Kontraktilität und Retraktion verantwortlich. Erst durch Koordination von Rac und Rho/ROCK entsteht somit Polarität und 3D mesenchymale Migration. / In the development from a primary tumor to metastatic dissemintation, which is the main cause of death from cancer, single cell migration through three-dimensional tumor stroma is an essential step. This work presents data concerning tumor cell migration and plasticity in a three-dimensional migration model. Small G-proteins control cytosceletal functions, especially actin polymerisation and the formation of cell protrusions through Rac as well as actomyosin contractility through Rho. Using pharmacological inhibitors of Rac and the Rho effector ROCK their impact on single-cell-migration in a three-dimensional model and particularly on morphology and plasticity of migration of tumor cells should be clarified. After inhibition of ROCK highly invasive HT1080 fibrosarcoma cells show a multipolar-dendritic and sessile phenotype. Inhibtion of Rac however induced a rounded phenotype which was also apolar and sessile. Simultaneous inhibition of ROCK and Rac resulted in rounded, apolar, sessile cells with abortive pseudopods. After disturbing the balance of ROCK and Rac by constitutive activation of ROCK, a divided population of cells developed, consisting of rounded, blebby cells and elongated cells. After sorting the cells according to their level of ß1-integrin expression, cells with low expression of integrins adopted a rounded type of migration with blebby dynamic protrusions, whereas cells with high integrin expression migrated in a elongated-mesenchymal way. Thus ROCK and Rac control together and simultaneously mesenchymal single cell migration. While Rac mediates protrusion, ROCK is responsible for contractility and retraction. Consequently only by coordination of Rho/ROCK and Rac polarity and mesenchymal 3D migration becomes possible.

Genetic and epigenetic factors that predispose to ineffective control of subgingival biofilm composition are incompletely understood. The objective of this study was to elucidate how leukocytes impact on the course of periodontitis in Rac-null mice. Models of acute gingivitis and periodontitis were used to assess the early inflammatory response and patterns of chronicity leading to alveolar bone loss. Leukocyte margination was differentially impaired during attachment in conditional Rac1-null and during rolling and attachment in Rac2-null mice. The inflammatory responses to subgingival ligatures were altered in Rac-null compared to WT mice. In response to persistent subgingival challenge Rac-null mice had increased alveolar bone loss with resorption patterns characteristic to aggressive periodontitis, partially explained by higher osteoclastic activity in Rac-null mice. This study demonstrates that migratory leukocyte defects are rate limiting steps in the periodontal inflammatory process that lead to more aggressive forms of periodontitis.

Genetic and epigenetic factors that predispose to ineffective control of subgingival biofilm composition are incompletely understood. The objective of this study was to elucidate how leukocytes impact on the course of periodontitis in Rac-null mice. Models of acute gingivitis and periodontitis were used to assess the early inflammatory response and patterns of chronicity leading to alveolar bone loss. Leukocyte margination was differentially impaired during attachment in conditional Rac1-null and during rolling and attachment in Rac2-null mice. The inflammatory responses to subgingival ligatures were altered in Rac-null compared to WT mice. In response to persistent subgingival challenge Rac-null mice had increased alveolar bone loss with resorption patterns characteristic to aggressive periodontitis, partially explained by higher osteoclastic activity in Rac-null mice. This study demonstrates that migratory leukocyte defects are rate limiting steps in the periodontal inflammatory process that lead to more aggressive forms of periodontitis.

One of the hallmarks of postprandial glucose homeostasis is the ability of insulin to promote glucose uptake into skeletal muscles. Insulin achieves this feat by enhancing the recruitment of glucose transporter 4 (GLUT4) from an intracellular compartment to the plasma membrane of muscles in order to create a net increase in surface GLUT4, which results in elevated glucose uptake. From a molecular perspective, this insulin-regulated GLUT4 traffic action requires the independent activation of Akt and Rac-1 in muscle cells because perturbation of either molecule results in an impaired response. Although Rac-1 has been validated as key component of insulin response, its downstream signalling capacity contributing to GLUT4 translocation remains unexplored. Studies on Rac-1 have shown that it is responsible for the formation of cortical remodelled actin that facilitates GLUT4 translocation following insulin stimulation. However, the downstream Rac-dependent molecules governing this actin remodelling are undetermined. Here we identified Arp2/3 and cofilin as the Rac-dependent regulators of insulin-induced actin remodelling in muscle cells. While Arp2/3 acts to initiate a burst of actin polymerization, cofilin balances out the actin dynamics through its severing/depolymerizing activity. Inhibition of either molecule’s function leads to defective GLUT4 translocation mediated by insulin in muscle cells, suggesting the requirement of actin dynamics to facilitate GLUT4 traffic to the plasma membrane. Furthermore, given the importance of Rac-1 in insulin-mediate GLUT4 traffic, its application potential to reverse insulin resistance has never been explored. We discovered that providing muscle cells with additional Rac-1 activity produces an insulin-independent gain in surface GLUT4 with magnitude comparable to that normally elicited by insulin. This phenotype is accomplished because of the concomitant cross-activation of Akt pathway when supplying the cells with active Rac-1. Interestingly, this response can bypass signalling defects imposed by cellular insulin resistance conditions, leading to restoration of GLUT4 translocation in muscle cells. Overall, these results not only reinforce the functional impact of Rac-1 on GLUT4 traffic but also identify additional molecules governed by Rac-1 contributing to the integrity of this insulin-mediated response in muscle cells.

Yes / Coronin7 (CRN7) stabilizes F-actin and is a regulator of processes associated with the actin cytoskeleton. Its loss leads to defects in phagocytosis, motility and development. It harbors a CRIB (Cdc42- and Rac-interactive binding) domain in each of its WD repeat domains which bind to Rac GTPases preferably in their GDP-loaded forms. Expression of wild type CRN7 in CRN7 deficient cells rescued these defects, whereas proteins with mutations in the CRIB motifs which were associated with altered Rac binding were effective to varying degrees. The presence of one functional CRIB was sufficient to reestablish phagocytosis, cell motility and development. Furthermore, by molecular modeling and mutational analysis we identified the contact regions between CRN7 and the GTPases. We also identified WASP, SCAR and PAKa as downstream effectors in phagocytosis, development and cell surface adhesion, respectively, since ectopic expression rescued these functions.

Thesis (Ph. D.)--University of Virginia, 2008. / Spine title: Characterization of BAI1. Includes bibliographical references. Also available online through Digital Dissertations as viewed 4/21/2009.

Endothelzellen kleiden als einschichtiger Zellverband das Innere der Blutgefäße aus und wirken als Barriere zwischen Blut und Interstitium. Entzündungen und Erkrankungen wie Lungenödem oder Arteriosklerose sind gekennzeichnet durch einen Zusammenbruch der Endothelbarriere. Erste Untersuchungen deuten auf eine bedeutende Rolle der GTPasen der Rho-Familie mit den Hauptvertretern Rho A, Rac 1 und Cdc42 als Regulatoren der Endothelbarriere hin. Bezüglich der Regulation der Endothelbarriereintegrität werden den GTPasen Rho A und Rac 1 meist antagonistische Funktionen zugeschrieben. In einem ersten Teil dieser Dissertation wurde daher die Funktion der Rho-GTPasen Rho A, Rac 1 und Cdc42 für die Endothelbarriere in verschiedenen Endothelien untersucht. Hierzu wurden drei mikrovaskuläre Endothelzelltypen verschiedenen Ursprungs sowie makrovaskuläre Endothelzellen der Pulmonalarterie mit GTPase-aktivierenden oder inaktivierenden bakteriellen Toxinen behandelt. Die Aktivierung von Rho A resultierte in allen Endothelzelltypen mit Ausnahme der mikrovaskulären myokardialen Endothelzellen in einem Zusammenbruch der Endothelbarriere. Die Aktivierung von Rac 1 und Cdc42 führte in allen Endothelzellarten zu einer Barrierestabilisierung. Darüber hinaus konnte in fast allen Endothelzelltypen durch pharmakologische Inhibition der Rho-Kinase eine Stabilisierung der Endothelbarriere induziert werden. Die Inaktivierung aller GTPasen sowie die alleinige Inaktivierung von Rac 1 führte zu einem kompletten Zusammenbruch der Endothelbarriere in vitro. Zudem ergaben in vivo-Experimente an perfundierten Rattenmesenterien eine gesteigerte Permeabilität nach Inaktivierung von Rho A, Rac 1 und Cdc42. Im zweiten Teil dieser Arbeit wurde die cAMP-vermittelte Stabilisierung der Endothelbarriere genauer charakterisiert und dabei der Einfluss gesteigerter cAMP-Spiegel auf die Aktivität von Rho-GTPasen in humanen dermalen mikrovaskulären Endothelzellen untersucht. Hierbei wurde die cAMP-Konzentration zum einen durch den Einsatz einer Kombination aus dem Adenylatzyklase-Aktivator Forskolin und dem Phosphodiesterase 4-Inhibitor Rolipram und zum anderen durch das cAMP-Analogon 8-pCPT-2’-O-Me-cAMP (O-Me-cAMP) gesteigert. O-Me-cAMP stellt hierbei einen selektiven Aktivator des cAMP nachgeschalteten Epac/Rap 1-Signalweges dar, wohingegen Forskolin/Rolipram durch die generelle cAMP-Steigerung zusätzlich die durch Proteinkinase A (PKA) vermittelten Signalwege stimuliert. Messungen des transendothelialen elektrischen Widerstandes zeigten nach cAMP-Anstieg in beiden Fällen eine Barrierestabilisierung, die mit den Effekten einer Aktivierung von Rac 1 vergleichbar waren. Dies ging mit Veränderungen der Organisation und der Morphologie von Zell-Zell-Kontakten einher. Zusätzlich kam es nach cAMP-Steigerung zu einer gesteigerten Rac 1-Aktivierung ohne Beeinflussung der Rho A-Aktivität. Darüber hinaus zeigten Endothelzellen nach cAMP-Anstieg die Bildung eines corticalen Aktinrings und verminderte Stressfaserbildung, was typische Indizien einer Aktivierung von Rac 1 sind. Um die Rolle von Rac 1 näher zu untersuchen, wurden Rac 1-Inhibitionsstudien durchgeführt. Die pharmakologische Inhibition der Rac 1-Aktivität resultierte in einer verminderten Endothelbarriereintegrität. Für beide cAMP-steigernden Mediatoren kann nach Kombinationsstudien angenommen werden, dass die durch cAMP-Steigerung vermittelten barrierestabilisierenden Effekte durch Rac 1 vermittelt zu sein scheinen. Somit kann aus den Untersuchungen des zweiten Teils dieser Arbeit geschlussfolgert werden, dass cAMP eine gesteigerte Endothelbarrierefunktion sowohl über PKA- als auch über Epac/Rap 1-abhängige Rac 1-Aktivierung vermittelt. Um die Rolle der Rho-GTPasen und von cAMP während einer Barrieredestabilisierung zu untersuchen, wurde im dritten Teil Thrombin als barrieredestabilisierender physiologischer Mediator in humanen dermalen mikrovaskulären Endothelzellen genutzt. Thrombin-Gabe führte zu einem reversiblen Zusammenbruch der Endothelbarriere. Zu diesen Zeitpunkten kam es zu einer signifikanten Inhibition von Rac 1 und einer deutlichen Aktivierung von Rho A. Erst nach 15 min fielen die gesamtzellulären cAMP-Spiegel ab. Innerhalb von 60 min erholte sich die Endothelbarriere und Rac 1- bzw. Rho A-Aktivitäten sowie der cAMP-Spiegel erreichten wieder ihr Ausgangsniveau. Vorinkubation der Endothelzellen mit beiden cAMP-steigernden Mediatoren inhibierte den Thrombin-induzierten Barriere-zusammenbruch ebenso wie die Thrombin-vermittelten Veränderungen der Rac 1- und Rho A-Aktivitäten. Auch in diesem Zusammenhang durchgeführte Rac 1-Inhibitionsstudien deuten darauf hin, dass die Hemmung der Thrombineffekte durch cAMP-Steigerung u.a. durch Aktivierung von Rac 1 vermittelt wird. / Endothelial cells build a monolayer coating the inner surface of blood vessels and thereby form a dynamic barrier between plasma and interstitial space. Impaired endothelial barrier function can result in vascular diseases such as edema, atherosclerosis and inflammation. Small GTPases of the Rho family such as Rho A, Rac 1 and Cdc42 are well known regulators of endothelial barrier integrity. It is generally believed that Rho A and Rac 1 regulate endothelial barrier functions in antagonistic manner. According to this concept, Rho A destabilizes barrier integrity whereas Rac 1 enhances endothelial barrier properties. In a first step we investigated the role of Rho A, Rac 1 and Cdc42 in endothelial barrier regulation in four different types of endothelial cells. Microvascular endothelial cells of different origin (myocardium, mesentery and dermis) and macrovascular endothelial cells from pulmonary artery were treated with bacterial toxins to specifically activate or inactivate Rho GTPases. Effects on endothelial barrier functions were revealed by immunfluorescence microscopy, measurement of transendothelial electrical resistance and FITC-dextran flux as well as by quantification of VE-cadherin-mediated adhesion using laser tweezers. Activation of Rho A resulted in break-down of endothelial barrier functions in all endothelial cell types except microvascular myocardial endothelial cells. Activation of Rac 1 and Cdc42 as well as pharmacological inhibition of Rho kinase stabilized endothelial barrier function in all endothelial cell types. Moreover, inactivation of all three GTPases as well as inactivation of Rac 1 alone resulted in endothelial barrier-breakdown in all endothelial cell types. From these data we conclude that Rac 1 is a highly important regulator required for maintenance of endothelial barrier function. In the second part of the study, we characterized the role of Rho GTPases in cAMP-mediated barrier stabilizing effects in microvascular endothelium. Therefore, we analyzed cAMP-induced effects on transendothelial electrical resistance, Rho GTPase activity and cell junction morphology in human dermal microvascular endothelial cells. To increase intracellular cAMP levels we used the cAMP-analogue 8-pCPT-2’-O-Me-cAMP (O-Me-cAMP) or combined treatment with adenylat cyclase-stimulating agent forskolin together with phosphodiesterase 4 inhibitor rolipram. In this approach O-Me-cAMP is used to selectively activate the Epac/Rap 1 pathway whereas forskolin/rolipram-induced increase of cAMP triggers both protein kinase A (PKA)- and Epac/Rap 1-dependent mechanisms. Measurement of transendothelial electrical resistance revealed barrier stabilizing effects of both Epac/Rap 1 and PKA signaling pathways. Barrier stabilization was accompanied by changes in cell junction morphology and both O-Me-cAMP and forskolin/rolipram treatment strongly activated Rac 1 without affecting Rho A activity. Moreover, endothelial cells displayed changes in actin distribution and cortactin localization typical for activation of Rac 1. To investigate the role of Rac 1 activation in cAMP-mediated barrier stabilization we performed Rac 1 inhibition studies. Pharmacological inhibition of Rac 1 activity decreased transendothelial electrical resistance which was accompanied by formation of intercellular gaps. Under these conditions the efficacy of increased cAMP to stabilize endothelial barrier functions was reduced and O-Me-cAMP had no effect. This indicates that barrier-stabilizing effects of cAMP are at least in part mediated by Rac 1-dependent mechanisms which are induced via PKA and Epac/Rap 1 signaling. Next, to address the importance of cAMP and of Rac 1 under conditions of impaired endothelial barrier integrity we used the physiological permeability-increasing mediator thrombin. Thrombin induced a transient breakdown of endothelial barrier function accompanied by increased stress fiber and gap formation in human dermal microvascular endothelial cells. Rac 1 was significantly inactivated whereas Rho A was strongly activated 5 and 15 min after thrombin treatment. Additionally, cAMP levels were decreased. After 60 min Rac 1 and Rho A activity as well as cAMP levels reached baseline values and endothelial barrier function was restored. Increase of cAMP completely blocked endothelial barrier breakdown and largely prevented thrombin-mediated effects on Rac 1 and Rho A activity indicating that reduction of cAMP was the primary mechanism causing the thrombin response. When Rac 1 was inactivated in parallel, both O-Me-cAMP and forskolin/Rolipram were not effective to prevent thrombin-induced endothelial barrier breakdown.

Recently, it was found that pathogens are trapped and killed by neutrophil extracellular traps (NETs). The role of Rac small GTPases is explored in the formation of NET using neutrophils lacking Rac1, Rac2 or both isoforms. NET formation was observed in both wild-type and Rac1-null neutrophils. In contrast, NET formation was markedly impaired in cells lacking either Rac2 or both Rac2 and Rac1. The defect in NET formation in Rac2-null cells was rescued in the presence of exogenous reactive oxygen species sources, suggesting that Rac2-mediated ROS generation is required. In addition, the role of nitric oxide in NET formation is assessed. Blocking NO production with the nitric oxide synthase inhibitor L-NAME significantly reduced NET formation. Moreover, Rac2-null cells produced significantly less NO than Rac1-null cells or their wild type counterparts. Our data suggest that Rac2 is essential for NET formation via pathways involving both ROS and NO.

Recently, it was found that pathogens are trapped and killed by neutrophil extracellular traps (NETs). The role of Rac small GTPases is explored in the formation of NET using neutrophils lacking Rac1, Rac2 or both isoforms. NET formation was observed in both wild-type and Rac1-null neutrophils. In contrast, NET formation was markedly impaired in cells lacking either Rac2 or both Rac2 and Rac1. The defect in NET formation in Rac2-null cells was rescued in the presence of exogenous reactive oxygen species sources, suggesting that Rac2-mediated ROS generation is required. In addition, the role of nitric oxide in NET formation is assessed. Blocking NO production with the nitric oxide synthase inhibitor L-NAME significantly reduced NET formation. Moreover, Rac2-null cells produced significantly less NO than Rac1-null cells or their wild type counterparts. Our data suggest that Rac2 is essential for NET formation via pathways involving both ROS and NO.