The role of podocytes in glomerular permselectivity is tightly associated with their intricate morphology, featuring interdigitating foot processes from adjacent cells. The actin cytoskeleton is an integral component of podocyte foot processes and is regulated by a number of proteins expressed in podocytes. Rho-family of small GTPases are known key regulators of the actin cytoskeleton. This study, investigated the role of Rac1 in podocytes, using conditionally immortalized mouse podocytes (MPs). We studied Rho-GTPase activities and morphology/cytoskeleton of differentiating mouse podocytes stably expressing nephrin. We also studied the impact of transfection of various Rho-GTPase mutants and IQGAP1 mutants. We demonstrated that nephrin expression potentiates and sustains Rac1 activity during the differentiation process. We showed that Rac1 contributes to process formation in differentiating MPs and may have a similar role in vivo when podocytes are recovering from injuries.

O carcinoma espinocelular de boca (CEB) é a neoplasia maligna mais comum da cavidade oral, correspondendo à aproximadamente 94% dos casos dessa região. Apesar dos diversos estudos moleculares e celulares do CEB, a taxa de sobrevida dos pacientes é de aproximadamente 50%, devido principalmente ao tamanho do tumor, metástase em linfonodos regionais, grau de diferenciação das células e sítio anatômico. O microambiente tumoral do CEB, é extremamente complexo e diversificado, tendo como característica principal um estado inflamatório crônico imunossupressivo. Este microambiente é sustentado pela liberação de diferentes citocinas inflamatórias, como IL-6, TNF- - atividades exercidas tanto pelas células tumorais quanto pelas estromais. Dentre essas atividades, tem sido relatado na literatura que as citocinas inflamatórias são capazes de aumentar a migração e a capacidade de invasão das células tumorais. Entre as células estromais, os macrófagos são as mais abundantes e participam da manutenção do microambiente tumoral. De acordo com o estímulo, podem ser polarizados M1, com papel pró-inflamatório e antitumoral, e M2, com papel anti-inflamatório e pró-tumoral. O objetivo desta tese foi compreender o papel dos macrófagos no prognóstico de CEB e das citocinas inflamatórias IL-6, TNF- - linhagens celulares de CEB. Para verificar o papel dos macrófagos no prognóstico, foi realizada uma revisão sistemática na qual foram incluídos apenas os estudos que utilizavam amostra de pacientes com CEB e avaliavam o prognóstico com marcadores para macrófagos. Foi observado que maiores concentrações de macrófagos CD68+ e CD163+ estavam relacionados com pior prognóstico de pacientes com CEB, embora não tenha sido possível concluir qual região tumoral a presença destas células seja mais importante 7 para o desfecho. Para analisar o papel das citocinas inflamatórias IL-6, TNFILensaios in vitro utilizando duas linhagens celulares, SCC25 e Cal27, em condições promotoras de migração sob a influência dessas citocinas. Foi observado que a citocina IL-6 foi capaz de aumentar a velocidade de migração e a direcionalidade tanto da SCC25 quanto da Cal 27 e que esta melhora na capacidade migratória ocorreu através de um crosstalk entre a via de sinalização relacionada a IL6 (STAT3) e a via reguladora de migração celular, Rho GTPase Rac1. Estes dados reforçam o papel do microambiente tumoral no processo de progressão tumoral e sugerem potenciais alvos terapêuticos como a modulação do perfil da população de macrófagos e o papel de interleucinas no controle de invasão tecidual e metástase. / Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity, corresponding to approximately 94% of the cases in this region. Despite the diverse molecular and cellular studies of OSCC, the patient survival rate is approximately 50%, mainly due to tumor size, regional lymph node metastasis, cell differentiation and anatomic site. The OSCC tumor microenvironment is extremely complex and diverse, with the main characteristic being an immunosuppressive chronic inflammatory state. This microenvironment is supported by the release of different inflammatory cytokines, such as IL-6, TNF- - and enhance the activities of both tumor and stromal cells. Among these activities, it has been reported in the literature that inflammatory cytokines are capable of increasing migration and invasiveness of tumor cells. Among stromal cells, macrophages are the most abundant and participate in the maintenance of the tumor microenvironment. According to the stimulus, macrophages can be polarized in M1, with pro-inflammatory and anti-tumoral role, and M2, with antiinflammatory and pro-tumoral role. Thus, the aim of this thesis was to evaluate the role of macrophages in the prognosis of OSCC and the influence of inflammatory cytokines IL-6, TNF- - OSCC cell lines. To assess the role of macrophages in the prognosis, a systematic review was conducted in which only studies using a sample of OSCC patients were evaluated and the prognosis was evaluated with macrophage markers. It was observed that higher concentrations of CD68 + and CD163 + macrophages were related to worse prognosis in patients with OSCC, although it was not possible to conclude which tumor region the presence of these cells is more important for the outcome. In order to analyze the role of the inflammatory cytokines IL-6, TNF- - atory 9 behavior of OSCC cells, in vitro assays using two cell lines, SCC25 and Cal27, were performed in migration-promoting conditions under the influence of these cytokines. It was observed that IL-6 was able to increase the speed migration and directionality of both SCC25 and Cal 27 and that this improvement in migratory capacity occurred through a crosstalk between the IL6-related signaling pathway (STAT3) and cell migration-related pathway, RhoGTPase Rac1. These data reinforce the role of the tumor microenvironment in the tumor progression process and suggest potential therapeutic targets such as the modulation of the profile of the macrophages population and the role of interleukins in the control of tissue invasion and metastasis.

O carcinoma espinocelular de boca (CEB) é a neoplasia maligna mais comum da cavidade oral, correspondendo à aproximadamente 94% dos casos dessa região. Apesar dos diversos estudos moleculares e celulares do CEB, a taxa de sobrevida dos pacientes é de aproximadamente 50%, devido principalmente ao tamanho do tumor, metástase em linfonodos regionais, grau de diferenciação das células e sítio anatômico. O microambiente tumoral do CEB, é extremamente complexo e diversificado, tendo como característica principal um estado inflamatório crônico imunossupressivo. Este microambiente é sustentado pela liberação de diferentes citocinas inflamatórias, como IL-6, TNF- - atividades exercidas tanto pelas células tumorais quanto pelas estromais. Dentre essas atividades, tem sido relatado na literatura que as citocinas inflamatórias são capazes de aumentar a migração e a capacidade de invasão das células tumorais. Entre as células estromais, os macrófagos são as mais abundantes e participam da manutenção do microambiente tumoral. De acordo com o estímulo, podem ser polarizados M1, com papel pró-inflamatório e antitumoral, e M2, com papel anti-inflamatório e pró-tumoral. O objetivo desta tese foi compreender o papel dos macrófagos no prognóstico de CEB e das citocinas inflamatórias IL-6, TNF- - linhagens celulares de CEB. Para verificar o papel dos macrófagos no prognóstico, foi realizada uma revisão sistemática na qual foram incluídos apenas os estudos que utilizavam amostra de pacientes com CEB e avaliavam o prognóstico com marcadores para macrófagos. Foi observado que maiores concentrações de macrófagos CD68+ e CD163+ estavam relacionados com pior prognóstico de pacientes com CEB, embora não tenha sido possível concluir qual região tumoral a presença destas células seja mais importante 7 para o desfecho. Para analisar o papel das citocinas inflamatórias IL-6, TNFILensaios in vitro utilizando duas linhagens celulares, SCC25 e Cal27, em condições promotoras de migração sob a influência dessas citocinas. Foi observado que a citocina IL-6 foi capaz de aumentar a velocidade de migração e a direcionalidade tanto da SCC25 quanto da Cal 27 e que esta melhora na capacidade migratória ocorreu através de um crosstalk entre a via de sinalização relacionada a IL6 (STAT3) e a via reguladora de migração celular, Rho GTPase Rac1. Estes dados reforçam o papel do microambiente tumoral no processo de progressão tumoral e sugerem potenciais alvos terapêuticos como a modulação do perfil da população de macrófagos e o papel de interleucinas no controle de invasão tecidual e metástase. / Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity, corresponding to approximately 94% of the cases in this region. Despite the diverse molecular and cellular studies of OSCC, the patient survival rate is approximately 50%, mainly due to tumor size, regional lymph node metastasis, cell differentiation and anatomic site. The OSCC tumor microenvironment is extremely complex and diverse, with the main characteristic being an immunosuppressive chronic inflammatory state. This microenvironment is supported by the release of different inflammatory cytokines, such as IL-6, TNF- - and enhance the activities of both tumor and stromal cells. Among these activities, it has been reported in the literature that inflammatory cytokines are capable of increasing migration and invasiveness of tumor cells. Among stromal cells, macrophages are the most abundant and participate in the maintenance of the tumor microenvironment. According to the stimulus, macrophages can be polarized in M1, with pro-inflammatory and anti-tumoral role, and M2, with antiinflammatory and pro-tumoral role. Thus, the aim of this thesis was to evaluate the role of macrophages in the prognosis of OSCC and the influence of inflammatory cytokines IL-6, TNF- - OSCC cell lines. To assess the role of macrophages in the prognosis, a systematic review was conducted in which only studies using a sample of OSCC patients were evaluated and the prognosis was evaluated with macrophage markers. It was observed that higher concentrations of CD68 + and CD163 + macrophages were related to worse prognosis in patients with OSCC, although it was not possible to conclude which tumor region the presence of these cells is more important for the outcome. In order to analyze the role of the inflammatory cytokines IL-6, TNF- - atory 9 behavior of OSCC cells, in vitro assays using two cell lines, SCC25 and Cal27, were performed in migration-promoting conditions under the influence of these cytokines. It was observed that IL-6 was able to increase the speed migration and directionality of both SCC25 and Cal 27 and that this improvement in migratory capacity occurred through a crosstalk between the IL6-related signaling pathway (STAT3) and cell migration-related pathway, RhoGTPase Rac1. These data reinforce the role of the tumor microenvironment in the tumor progression process and suggest potential therapeutic targets such as the modulation of the profile of the macrophages population and the role of interleukins in the control of tissue invasion and metastasis.

The endothelial cells lining the inner surface of the tissue capillaries and post-capillary venules form a semi-permeable barrier between the blood circulation and interstitial compartments. The semi-permeable barrier in these vessels is the major site of blood-tissue exchange. A compromised endothelial barrier contributes to the pathological process such as edema, acute respiratory distress syndrome (ARDS) and tumor metastasis. Sphingosine-1-phosphate (S1P), an endogenous, bioactive lipid present in all cells, is a potential therapeutic agent that can restore compromised endothelial barrier function. On the other hand, S1P also has pleotropic effects and can either increase or decrease arterial tone and tissue perfusion under different conditions. The detailed mechanisms underlining S1P’s endothelial barrier protective effect are still largely unknown, but are suggested to depend on cell spreading termed “lamellipodia”. Therefore, to fully take advantage of the beneficial properties of S1P, it is important to first understand how S1P-induced lamellipodia protrusions correlate with its effect on endothelial barrier function. It is also important to know the underlining mechanisms that S1P enhances endothelial barrier function, including intracellular signaling and receptor signaling. To study local lamellipodia activities, we acquired time-lapse images of live endothelial cells expressing GFP-actin, and subsequently analyzed different lamellipodia parameters. Experiments were performed under baseline conditions, and during endothelial barrier disruption or enhancement. The compounds used in these experiments included thrombin and S1P. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function for in vitro studies. Changes of local lamellipodia dynamics and endothelial barrier function within the same time frame were studied. For mechanistic studies, we combined biochemical, immunological and pharmacological approaches. Rho family small GTPase activities were measured with an ELISA pull-down assay. Fluorescence Resonance Energy Transfer (FRET) was also used to study the localization of RhoA activation. Pharmacological compounds targeting intracellular signaling messengers were used to test the involvement of Rac1, RhoA, MLC-2 in endothelial local lamellipodia activity and S1P-mediated endothelial barrier enhancement. Receptor agonists and antagonists were used to study the involvement of S1P receptor signaling. Finally, for cell behavior and cytoskeleton studies, we utilized immunofluorescence labeling that enables direct visualization of changes in cytoskeleton, cell-cell junction and focal adhesions. We found that S1P increases both local lamellipodia protrusions and TER. The rapid increase in local lamellipodia protrusion frequencies also corresponded to the rapid increase in TER seen within the same time frame. Under the microscope, local lamellipodia protrusions from adjacent cells overlapped with each other and extended beyond junctional cell-cell contacts. Strikingly, S1P-induced lamellipodia protrusions carry VE-cadherin molecules to the cell-cell contact, established junctional adhesions. Combined with our previous published studies on thrombin induced lamellipodia activity changes, we think lamellipodia protrusions are a major component that regulates endothelial barrier function. Combined, our imaging studies revealed the mechanisms on how lamellipodia regulates endothelial barrier function: 1) lamellipodia overlap and increase the apical to basal diffusion distance, which in turn decreases permeability and upregulates endothelial barrier function. 2) Local lamellipodia protrusions contain VE-cadherin, which is delivered the to the cell-cell contact by the lamellipodia to increase junctional stability. S1P is effective for rescuing thrombin-induced endothelial barrier dysfunction. The known barrier disruptor thrombin, decreased local lamellipodia protrusions, disrupted VE-cadherin integrity, and caused a drop in TER. S1P increased local lamellipodia protrusions after thrombin challenge, and resulted in faster recovery towards baseline TER compared with vehicle controls. Interestingly, we also found that both thrombin and S1P increased MLC-2 phosphorylation at Thr18/Ser19. We subsequently accessed Rho family GTPase activity after thrombin and S1P. As expected, thrombin rapidly increased GTP-bound RhoA levels, and decreased GTP-bound Rac1 levels. Unexpectedly, S1P not only increased GTP-bound Rac1, but also increased GTP-bound RhoA to a more prominently levels (4-fold). Since Rac1 has been implicated in promoting lamellipodia protrusions, we tested the role of Rac1 on the local lamellipodia activities first. We found that Wild-Type (WT) Rac1 group had the highest local lamellipodia protrusion frequencies, protrusion distances, withdraw time and highest percentage of protrusions that lasted more than 5 min. WT Rac1 overexpression had greatest protrusion frequencies and lowest monolayer permeability to FITC-albumin compared to GFP and DN-Rac1 overexpression monolayers. These results suggest that Rac1 is important for baseline endothelial barrier function. This is also confirmed by the finding that pharmacological inhibition of Rac1 significantly decreased baseline TER. Although Rac1 is important for baseline endothelial barrier function, we noticed that it is dispensable in S1P-mediated endothelial barrier enhancement. Rac1 inhibitors, DN-Rac1 overexpression, and Rac1 siRNA knockdown all failed to abolish the S1P-mediated increase in TER. This is partially explained by the findings that S1P-induced Rac1 activation is short-lived and less pronounced in contrast to RhoA activation. We subsequently tested the role of RhoA in S1P-mediated endothelial barrier enhancement, based on our findings that both S1P and thrombin significantly activated RhoA and induced MLC-2 phosphorylation. Significant RhoA activation was found to be mainly at cell periphery and lamellipodia protrusions in HUVEC on FRET, after S1P was given. In addition, RhoA inhibitors significantly decreased the amplitude of S1P-induced MLC-2 phosphorylation, vinculin redistribution and barrier enhancement. The data suggest that the mechanisms involved in S1P-mediated endothelial barrier enhancement depend on RhoA activation and subsequent cytoskeletal rearrangement. We next investigated which receptor is responsible for the endothelial barrier enhancement of S1P. However, antagonism of S1P1, S1P2 or S1P3 alone with W146, JTE-013 or TY-52156 respectively all failed to attenuate S1P-mediated increase in TER. While agonism of S1P1 with CYM-5442 hydrochloride alone produced significant increase in TER, neither S1P2 nor S1P3 activation (CYM 5520 & CYM 5541) produced any change on TER. Interestingly, S1P1 antagonist failed to block the effect of S1P1 agonist on TER. This could be due to that the S1P1 agonist may not be very selective at concentrations tested. We also identified that S1P4 and S1P5 are present on endothelial cells. Further studies would be necessary to elucidate the roles of newly identified S1P4 or S1P5 alone on endothelial barrier function. It is also worth investigating in the future if multiple S1P receptors are involved in its endothelial barrier enhancing effect. In conclusion, we found that lamellipodia protrusions contribute to the endothelial barrier enhancement of S1P. While Rac1 is important for the maintenance of endothelial barrier function, it is dispensable in S1P-mediated endothelial barrier enhancement. On the other hand, RhoA activation appears to be, at least in part, responsible for the endothelial barrier enhancement of S1P. It is currently still unclear if S1P’s endothelial barrier enhancing effect is through one single receptor activation or activation of multiple receptors. Future studies are needed to elucidate the receptor signaling that contributes to S1P-mediated endothelial barrier enhancement.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18455号 / 医博第3910号 / 新制||医||1004(附属図書館) / 31333 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 小川 誠司, 教授 宮本 享 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The classical description of the innate antiviral response involves the production of type I interferon (IFN) and the subsequent expression of hundreds of interferon stimulated genes (ISGs), which cooperatively repress viral replication and spread. More recently, an IFN-independent antiviral response has also been described, in which the entry of an enveloped virus induces a subset of ISGs without requiring the production of IFN, although the details of this response remain unclear. In this work, multiple approaches were used to further characterize antiviral signaling pathways. Initially, the potential involvement in the IFN-independent response of the small GTPase Rac1, which has been implicated in both viral entry and antiviral signaling, was investigated. Here, Rac1 was shown to have a possible function in the negative regulation of ISG expression, although technical complications prevented definitive conclusions. As an alternative strategy to identify novel aspects of antiviral signaling, the mechanism of action of ICP0, a herpes simplex virus (HSV) protein involved in innate immune evasion, was investigated. Although ICP0 is generally thought to perform its actions in the nucleus, by tagging proteins for proteasome-mediated degradation via the E3 ubiquitin ligase activity of its RING finger domain, here it was shown that not only does cytoplasmic ICP0 have a RING-dependent but proteasome-independent ability to block antiviral signaling, but also that ICP0 has a previously unknown RING-independent function in the promotion of viral replication in the cytoplasm. To further investigate the cytoplasmic activities of ICP0, proteins interacting with ICP0 in the cytoplasm were identified using quantitative mass spectrometry. This revealed several intriguing binding partners for ICP0, including WDR11, a poorly-characterized cellular protein which was shown to undergo a dramatic relocation during HSV infection, although it was not required for viral replication in cultured cells. Therefore, this study has uncovered several new and unexpected insights into ICP0 behavior. / Thesis / Doctor of Philosophy (PhD)

Aims: Oxidative stress and inflammation contribute to atherogenesis. Rac1 GTPase regulates pro-oxidant NADPH oxidase activity, reactive oxygen species (ROS) formation, actin cytoskeleton organization and monocyte adhesion. We investigated the vascular effects of pharmacological inhibition of Rac1 GTPase in mice. Methods and Results: We treated wild-type and apolipoprotein E-deficient (ApoE−/−) mice with Clostridium sordellii lethal toxin (LT), a Rac1 inhibitor, and assessed vascular oxidative stress, expression and activity of involved proteins, endothelial function, macrophage infiltration, and atherosclerosis development. LT-treated wild-type mice displayed decreased vascular NADPH oxidase activity and ROS production. Therapeutic LT doses had no impact on behavior, food intake, body weight, heart rate, blood pressure, vascular and myocardial function, differential blood count, and vascular permeability. ApoE−/− mice were fed a cholesterol-rich diet and were treated with LT or vehicle. LT treatment led to decreased aortic Rac1 GTPase activity, NADPH oxidase activity and ROS production, but had no impact on expression and membrane translocation of NADPH oxidase subunits and RhoA GTPase activity. LT-treated mice showed improved aortic endothelium-dependent vasodilation, attenuated atherosclerotic lesion formation and reduced macrophage infiltration of atherosclerotic plaques. Concomitant treatment of cholesterol-fed ApoE−/− mice with LT, the specific synthetic Rac1 inhibitor NSC 23766 or simvastatin comparably reduced aortic Rac1 activity, NADPH oxidase activity, oxidative stress, endothelial dysfunction, atherosclerosis development, and macrophage infiltration. Conclusions: These findings identify an important role of the small GTPase Rac1 in atherogenesis and provide a potential target for anti-atherosclerotic therapy.

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