Modulation of the actin-based cytoskeleton and transcription factor regulation are merely two essential functions in a wide array of cellular activities that the Rho family of small GTPases is responsible for mediating. Aberrations in, or loss of, Rho GTPase signaling has been found to lead to multiple pathologies, including both metastatic progression and lenticular differentiation leading to cataractogenesis. This study has examined the transcriptional regulation of the metastasis suppressor, KiSS-1. Although the mechanism by which KiSS-1 modulates an anti-metastatic effect is not entirely known, it is known that KiSS-1 mediates stress fiber formation, increased adhesion and reduced migratory and invasive properties through modulation of the Rho family of small GTPases. The loss of KiSS-1 that commonly occurs during metastatic progression, leads to a loss of proper Rho GTPase regulation. This study has examined how KiSS-1 is regulated in two tissue types, breast and skin, and how the loss of AP-2(alpha) and DRIP-130, respectively, leads to the progression of breast cancer and melanoma. In addition, this study has also looked at the importance of Rac1 expression and function in the lens epithelium. Activation of Rac1 and its downstream effector, SRF, have been shown to be key regulators in lens cell differentiation, possibly leading to lens opacification via its transcriptional control of the structural crystallins within the lens. The results of this dissertation research have made significant strides in understanding the nature of the anti-metastatic effects registered by the novel KiSS-1 peptide and its cognate GPCR. Additionally, it has shed light on the Rho family regulation of lens epithelial cell differentiation, indicating the elaborate involvement of Rac1 in mediating lens fiber development. In all, this research has determined previously unknown roles of small molecule GTPases in both the progression of metastasis, as well as in normal and abnormal lens cell differentiation.

Modulation of the actin-based cytoskeleton and transcription factor regulation are merely two essential functions in a wide array of cellular activities that the Rho family of small GTPases is responsible for mediating. Aberrations in, or loss of, Rho GTPase signaling has been found to lead to multiple pathologies, including both metastatic progression and lenticular differentiation leading to cataractogenesis. This study has examined the transcriptional regulation of the metastasis suppressor, KiSS-1. Although the mechanism by which KiSS-1 modulates an anti-metastatic effect is not entirely known, it is known that KiSS-1 mediates stress fiber formation, increased adhesion and reduced migratory and invasive properties through modulation of the Rho family of small GTPases. The loss of KiSS-1 that commonly occurs during metastatic progression, leads to a loss of proper Rho GTPase regulation. This study has examined how KiSS-1 is regulated in two tissue types, breast and skin, and how the loss of AP-2(alpha) and DRIP-130, respectively, leads to the progression of breast cancer and melanoma. In addition, this study has also looked at the importance of Rac1 expression and function in the lens epithelium. Activation of Rac1 and its downstream effector, SRF, have been shown to be key regulators in lens cell differentiation, possibly leading to lens opacification via its transcriptional control of the structural crystallins within the lens. The results of this dissertation research have made significant strides in understanding the nature of the anti-metastatic effects registered by the novel KiSS-1 peptide and its cognate GPCR. Additionally, it has shed light on the Rho family regulation of lens epithelial cell differentiation, indicating the elaborate involvement of Rac1 in mediating lens fiber development. In all, this research has determined previously unknown roles of small molecule GTPases in both the progression of metastasis, as well as in normal and abnormal lens cell differentiation.

No description available.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23334号 / 生博第452号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 永尾 雅哉, 教授 渡邊 直樹, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

L'épithélium intestinal se renouvelle tous les 4 à 6 jours chez les mammifères grâce aux cellules souches localisées au fond des cryptes. Le renouvellement dépend des signaux émis par le microenvironnement et requiert une phase de prolifération des cellules souches, de différenciation et d'apoptose/desquamation des cellules épithéliales. La signalisation Wnt, joue un rôle majeur dans l'homéostasie intestinale par l'action de deux gradients inversés le long de l'axe crypte/lumière ; la signalisation Wnt canonique, active au fond des cryptes, contrôle la prolifération alors que la signalisation non-canonique, active vers le haut des cryptes contrôle la différenciation. Il a été montré que ces deux voies contrôlent l'activité de la GTPase atypique RhoU/Wrch1. RhoU fait partie des GTPases qui s'activent spontanément, son activité est donc directement proportionnelle à son niveau d'expression dans la cellule. Enfin, cette GTPase atypique est sous exprimée dans de nombreuses tumeurs gastriques et colorectales.Compte tenu de ces données, nos objectifs étaient donc de caractériser les changements morphologiques induits par l'invalidation conditionnelle de RhoU dans l'épithélium intestinal murin et d'en déterminer les mécanismes d'action. Nos résultats montrent que la déplétion de RhoU n'est pas létale, cependant elle a induit une augmentation de 20% de la densité cellulaire et une désorganisation de la structure de l'épithélium dans le haut des cryptes du colon. Cette augmentation concerne aussi bien les lignages sécrétoires et absorptifs, cependant, l'absence de RhoU a induit une sur-représentation du lignage sécrétoire. Dans la lignée de tumeur colorectale DLD-1, nous avons montré que l'absence de RhoU mime le phénotype d'augmentation de la densité cellulaire observé chez la souris. L'invalidation de RhoU ne modifie pas la distribution des phases du cycle cellulaire ni de celle de la mitose, cependant, elle réduit le nombre des cellules en apoptose dans le colon des souris et dans les cellules DLD-1. L'invalidation de RhoU a réduit la signalisation Hippo et a altéré la contractilité cellulaire via une augmentation de la phosphorylation de la protéine MLC2. Des travaux récents ont montré que la diminution du niveau MLC2 phosphorylée est nécessaire pour l'activation des caspases par un stimulus apoptotique. Ceci suggère que cette perturbation de la contractilité peut être à l'origine de cette diminution de l'apoptose qui est la cause majeure responsable de ce phénotype. En conclusion, RhoU est un régulateur de l'homéostasie intestinale chez la souris via son rôle modérateur de la mort cellulaire. / In Mammals, the intestinal epithelium is renewed every 4-6 days through the stem cells located at the bottom of crypts. The renewal depends on signals from the micro-environment and requires a proliferation phase of stem cells, then a differentiation and apoptosis/desquamation phases of epithelial cells. Wnt signaling plays a major role in intestinal homeostasis by the action of two reversed gradients along the axis crypt/ lumen: canonical Wnt signaling, active in the bottom of crypts, control proliferation while non canonical signaling, active in the top of the crypts control cell differentiation. It was shown that these two pathways are regulator of the atypical GTPase RhoU/Wrch1. The RhoU protein activates spontaneously, its activity is directly proportional to its expression level in the cell and is expressed as in gastric and colorectal tumors. In view of these informations, our objectives were therefore to characterizethe morphological changes induced by conditional invalidation of RhoU in the intestinal epithelium of mice and to determine the mechanisms of action. Our results show that RhoU depletion is not lethal. However, it induces an increase of cell density (+20%) and a disruption of the epithelium structure in the top of the colonic crypts. This increase affects both absorptive and secretory lineages. However, the absence of RhoU induced over-representation of secretory lineage. In colorectal tumor cell line DLD-1, we have shown that the absence of RhoU mimics the phenotype of cell density increase observed in mice. RhoU invalidationdid not change the distribution of cell cycle phases and mitosis, however, it reduces the number of apoptotic cells in the colon of mice and in the DLD-1 cells. RhoU invalidation reduced Hippo signaling and altered cell contractility via the increase of the protein MLC2 phosphorylation. Recent work has shown that the reduction of MLC2-P level is necessary for the caspase protein activation by an apoptotic stimulus. Suggesting that the perturbation of contractility may be the cause of this apoptosis decrease which is the main cause responsible of this phenotype. Finally, RhoU is a regulator of the intestinal homeostasis in micevia its moderating role of cell death.

Dynamic regulation of the actin cytoskeleton is required for synapses to form and maintain their shape. The actin cytoskeleton is regulated by Rho GTPases in response to genetic and extracellular signals. Rho GTPases are regulated by guanine nucleotide exchange factors and GTPase activating proteins (GAPs). Syd-1 is a protein that has been identified as necessary for synapse formation in worms, with similar proteins in flies, and mice. Little is known about the molecular mechanism by which Syd-1 is acting. While genetic techniques are great tools for examining synapse development, they are limited by their inability to consider the molecular nature of the protein product. By studying the biochemical nature of synaptic proteins, we can begin to understand their function with a new level of clarity. Syd-1 has a predicted Rho GAP domain; however it is thought to be inactive. The activity of the fly protein, Dsyd-1, has never been examined although it has been speculated that it is inactive in all invertebrates. Recently the mouse version was reported to have Rho GAP activity. By performing GTPase activity assays on purified proteins, I found the GAP domain of Dsyd-1 increased the GTPase activity of Rac-1 and Cdc42 but not RhoA. Members of our lab found the activity of Dsyd-1 is necessary for proper synapse formation both at the Drosophila neuromuscular junction as well as in R7 neurons. In Caenorhabditis elegans, Syd-1 was found to interact with presynaptic protein RSY-1. Since RSY-1 is evolutionarily conserved, I tested whether or not RSY-1 has a similar effect on R neurons in Drosophila. I also isolated mRNA from R neurons and evaluated the possibility of analyzing mutant neurons using comparative transcriptomics. This dissertation includes previously unpublished coauthored material.

Zwischen Blutkompartiment und umliegenden Interstitium besteht eine Barriere, die durch eine einzelne Schicht aus Endothelzellen gebildet wird. Essentiell für diese Barriere, deren Funktion in der Begrenzung des Austausches von Flüssigkeit und gelösten Stoffen liegt, sind interzelluläre Junktionen, welche die Endothelzellen miteinander verbinden. Durch eine gestörte Funktion und Regulation der Endothelbarriere entstehen beim Menschen verschiedene Pathologien wie zum Beispiel Ödeme, hämorrhagischer Schlaganfall und vaskuläre Malformationen. Es ist bekannt, dass cAMP die Endothelbarriere zum Teil durch Aktivierung der kleinen GTPase Rac1 stabilisiert. Trotz der großen medizinischen Relevanz dieses Signalweges, sind die damit einhergehenden Effekte auf die interzellulären Kontakte auf ultrastruktureller Ebene weitgehend unbekannt. In mikrovaskulären Endothelzellkulturen kam es ähnlich wie in intakten Mikrogefäßen zur Stärkung der Barrierefunktion. So resultierte sowohl nach Behandlung mit Forskolin und Rolipram (F/R), welche zur Steigerung der intrazellulären cAMP-Spiegel führen, als auch nach Zugabe von 8-(4-chlorophenylthio)-2´-O-methyladenosin-3´,5´-cyclic monophosphorothioate (O-Me-cAMP), einem selektiven Aktivator des cAMP nachgeschalteten Epac/Rap1-Signalweges, ein Anstieg des TER; außerdem konnte durch beide Substanzen (F/R und O-Me-cAMP) die Aktivierung von Rac1 induziert werden. Desweiteren wurde eine verstärkte Intensität und Linearisierung des Immunfluoreszenzsignals der Zelljunktionsproteine VE-Cadherin und Claudin5 entlang der Zellgrenzen beobachtet. In der ultrastrukturellen Analyse der interzellulären Kontaktzonen-Architektur zeigte sich unter F/R- oder O-Me-cAMP-Exposition ein signifikanter Anstieg an komplexen Interdigitationen. Diese komplexen Strukturen waren dadurch charakterisiert, dass sich die Membranen benachbarter Zellen, die durch zahlreiche endotheliale Junktionen stabilisiert wurden, über vergleichsweise lange Distanzen eng aneinanderlegten, so dass ein deutlich verlängerter Interzellularspalt resultierte. Die Inhibition der Rac1-Aktivierung durch NSC-23766 verminderte die Barrierefunktion und blockierte effektiv die O-Me-cAMP-vermittelte Barrierestabilisierung und Reorganisation der Kontaktzone einschließlich der Junktionsproteine. Demgegenüber konnte die F/R-vermittelte Barrierestabilisierung durch NSC-23766 nicht beeinträchtigt werden. Parallel dazu durchgeführte Experimente mit makrovaskulären Endothelien zeigten, dass es in diesem Zelltyp unter Bedingungen erhöhter cAMP-Konzentrationen weder zur Rac1-Aktivierung noch zur Barrierestärkung oder Kontaktzonen-Reorganisation kam. Diese Ergebnisse deuten darauf hin, dass in mikrovaskulären Endothelien Rac1-vermittelte Änderungen der Kontaktzonen-Morphologie zur cAMP-induzierten Barrierestabilisierung beitragen. / Evidence exists that cAMP stabilizes the endothelial barrier in part via activation of the small GTPase Rac1. However, despite the high medical relevance of this signaling pathway, the mechanistic effects on intercellular contacts on the ultrastructural level are largely unknown. In microvascular endothelial cell monolayers, in which increased cAMP strengthened barrier properties similar to intact microvessels in vivo, both forskolin and rolipram (F/R) to increase cAMP and 8-(4-chlorophenylthio)-2´-O-methyladenosine-3´,5´-cyclic monophosphorothioate (O-Me-cAMP) to stimulate exchange protein directly activated by cAMP/Ras proximate-1 (Epac/Rap1) signaling enhanced transendothelial electrical resistance (TER) and induced activation of Rac1. Concurrently, augmented immunofluorescence intensity and linearization of signals at cell borders were observed for intercellular junction proteins VE-cadherin and claudin5. Ultrastructural analysis of the intercellular contact zone morphology documented that exposure to F/R or O-Me-cAMP led to a significant increase in the proportion of contacts displaying complex interdigitations of cell borders in which membranes of neighboring cells were closely apposed over comparatively long distances and which were stabilized by numerous intercellular junctions. Interference with Rac1 activation by NSC-23766 completely abolished both barrier stabilization and contact zone reorganization in response to O-Me-cAMP whereas F/R-mediated barrier enhancement was not affected by NSC-23766. In parallel experiments using macrovascular endothelium, increased cAMP failed to induce Rac1 activation, barrier enhancement and contact zone reorganization. These results indicate that in microvascular endothelium Rac1-mediated alterations in contact zone architecture contributes to cAMP-induced barrier stabilization.

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors, characterized by a highly invasive cell population. GB tumors develop treatment resistance and ultimately recur; the median survival is nearly fifteen months and importantly, the invading cell population is attributed with having a decreased sensitivity to therapeutics. Thus, there remains a necessity to identify the genetic and signaling mechanisms that promote tumor spread and therapeutic resistance in order to develop new targeted treatment strategies to combat this rapidly progressive disease. TWEAK-Fn14 ligand-receptor signaling is one mechanism in GB that promotes cell invasiveness and survival, and is dependent upon the activity of multiple Rho GTPases including Rac1. Here, we show that Cdc42 is essential in Fn14-mediated Rac1 activation. We identified two guanine nucleotide exchange factors (GEFs), Ect2 and Trio, involved in the TWEAK-induced activation of Cdc42 and Rac1, respectively, as well as in the subsequent TWEAK-Fn14 directed glioma cell migration and invasion. In addition, we characterized the role of SGEF in promoting Fn14-induced Rac1 activation. SGEF, a RhoG-specific GEF, is overexpressed in GB tumors and promotes TWEAK-Fn14-mediated glioma invasion. Moreover, we characterized the correlation between SGEF expression and TMZ resistance, and defined a role for SGEF in promoting the survival of glioma cells. SGEF mRNA and protein expression are regulated by the TWEAK-Fn14 signaling axis in an NF-kB dependent manner and inhibition of SGEF expression sensitizes glioma cells to TMZ treatment. Lastly, gene expression analysis of SGEF depleted GB cells revealed altered expression of a network of DNA repair and survival genes. Thus TWEAK-Fn14 signaling through the GEF-Rho GTPase systems which include the Ect2, Trio, and SGEF activation of Cdc42 and/or Rac1 presents a pathway of attractive drug targets in glioma therapy, and SGEF signaling represents a novel target in the setting of TMZ refractory, invasive GB cells.