Ligand-activated signal transduction is a process critical to cell survival and function as it serves as a means of communication between the cells and their environment. Endocytosis is generally thought to down-regulate incoming signals by reducing the surface availability of receptors. However, increasing evidence in many systems suggests a notion which is referred to as the „signalling endosome" hypothesis - that endocytosis can also actively contribute to signalling apart from clearance of activated receptors and thereby attenuation of signalling. The functional aspect of signalling endosomes has been well-characterized in several pathways including RTK and TGF-β signalling. There are, however, various other signalling pathways where the active mechanism of endocytotic regulation is yet to be understood. In this study, we probe this aspect in the cytokine signalling system, where the receptors are known to internalize but the significance of such internalization and precise mechanism is unclear. My thesis aims to elucidate the function and molecular details of internalization of cytokine receptor using interleukin-4 receptor (IL-4R) signalling as a model. IL-4 and IL-13 ligands can induce assembly of three distinct complexes: IL4 induced IL-4Rα – IL-2Rγ (type I), IL-4 induced IL-4Rα – IL-13Rα1 (type II) or the IL-13 induced IL-13Rα1-IL-4Rα (type II). The formation of any of these complexes triggers signalling through the JAK/STAT pathway. However, models of how the oligomerization of the transmembrane receptors and activation takes place are very diverse and lack a clear molecular and biophysical understanding of the underlying receptor dynamics. Previous results of the lab had shown that the affinities between subunits are low, precluding complex formation at the plasma membrane at physiological concentrations. In addition, IL-4R subunits localize in to endosomal structures adjacent to the plasma membrane. It had already been shown that the shared IL-4R subunit IL-2Rγ is internalized by a specific, actin dependent, Rac1/Pak1 regulated endocytosis route in the IL-2 context. We could show that pharmacological suppression of this endocytosis pathway also prevented IL-4 induced JAK/STAT signalling, placing endocytosis upstream of signalling. Here I show using immuno-EM techniques that these endosomal structures are multivesicular bodies. Importantly, I could show that receptor subunits are highly enriched in the limiting membrane of these endosomes relative to the adjacent plasma membrane. Using quantitative loading assays I could furthermore demonstrate that this enrichment is achieved by constitutive internalization of receptors from the cell surface into cortical endosomes. The trafficking kinetics of the receptor subunits is independent of ligand occupancy. Pharmacological inhibition shows that receptors and ligand traffic via the previously identified Rac1/Pak1 pathway. Finally, Vav2 was identified as a candidate Guanine Exchange Factor (GEF) that may regulate Rac1 activity and thereby control the actin polymerization cascade driving IL-4R endocytosis. Immunoprecipitations showed that Vav2 interacts both with the cytoplasmic tail region of the receptors and the receptor associated 2 kinase JAK3. Vav2 may thus couple the receptor/JAK complexes to the Rac1/Pak1 mediated endocytosis route. Taken together, our results suggests that stable „signalling endosomes‟ adjacent to the plasma membrane act as enrichment centres, where ligand and receptor concentrations are locally increased by constitutive trafficking. The confined environment of the endosome then compensates for the weak affinities between the ligand and receptor and facilitates ligand-mediated receptor dimerization. Importantly, overexpression of both type II IL-4R subunits renders signal transduction resistant to endocytosis inhibition, strongly suggesting that the critical factor effecting signalling is sufficient concentration, which the endosomes facilitate achieving. The endosomes are thus dispensable as signalling scaffolds when the receptors are in sufficient concentration, where activated receptors could interact with downstream pathway components. Endocytosis thus provides a crucial means for the signalling process to overcome the thermodynamic hurdles for receptor oligomerization. In conclusion, our data propose a novel, purely thermodynamic role of endosomes in regulating cytokine receptor signalling not seen in any other signalling pathway.

The JAK-STAT pathway is an intracellular signaling pathway that is found to have crucial roles in hematopoiesis, immune response and the development of many other tissues in mammals. The pathway is conserved in Drosophila melanogaster, and is much simpler: there is only one Drosophila JAK (Hopscotch, Hop) and STAT (STAT92E) respectively, while there are at least 4 JAKs and 7 STATs in mammals. The pathway has been intensively studied in Drosophila, and has been implicated in many tissue development and cellular processes. In this work, I present several roles of JAK signaling in oogenesis.First, JAK signaling is required for cell differentiation within a specific lineage of follicle cells – stalk cells and polar cells. Unpaired (upd), which encodes the known ligand for the pathway, is expressed specifically in the polar cells in the developing egg. Reduced function of Upd or Hop results in fusions of egg chambers, which is primarily caused by improper formation of stalk cells, while general activation of the pathway in the egg chamber produces an extra number of stalk cells and sometimes eliminates polarfollicle cells. Based on the known function of the Notch pathway in oogenesis, we propose a model that Notch signaling determines a pool of precursors for the polar and stalk cells while JAK activity determines their specific fates within that pool.Second, JAK signaling is also involved in epithelial follicle cell differentiation. Consistent with the expression pattern of upd in the ovary, there is a gradient of JAK activity expanding from the poles, and this JAK activation gradient is both required and sufficient to suppress the main body follicle cell fate. Also, different levels of JAK activity are required and sufficient to determine both anterior and posterior terminal follicle cell fates. Consistent with these data is a model that a gradient of JAK activity triggered by Upd from the poles pre-patterns the epithelium into three domains and pre-determines sub-populations of terminal follicle cell fates prior to the EGFR activation, and cooperates with EGFR activity later to define posterior terminal follicle cell fates. This provides the first evidence for a morphogenic function of the JAK-STAT pathway in any organism.

The JAK/STAT pathway is a non-receptor tyrosine kinase signaling pathway that is well conserved and highly re-utilized in many mammalian and Drosophila developmental processes. Compared to dozens of ligands and receptors in mammalian JAK/STAT, Drosophila JAK/STAT pathway is simpler with one receptor and three ligands, Upd, Upd2 and Upd3, which have similar amino acid sequences. Previous literature shows that upd and upd2 exhibit the same dynamic striped expression pattern in embryos and have semi-redundant functions during embryogenesis. Do Upd and Upd3 also have redundant functions? To answer this question, the functions of Upd3 in Drosophila development were investigated in this dissertation. In addition, the coordinate expression mechanism of upd and upd3 in eye discs was also analyzed. To study the functions of Upd3 in development, the expression pattern of upd3 was examined and detected in larval eye discs, wing discs, haltere discs, lymph glands and adult ovaries with in situ hybridization to upd3 mRNA and an upd3 reporter line. Consistent with the expression pattern, the loss of function mutants of upd3 exhibit small eyes, outstretched wings, downward extended halteres and reduced circulating blood cell concentration, demonstrating the roles of Upd3 in these tissues’ development. However, functions of Upd3 in other aspects of immune response were not detected. To investigate the mechanism of the coordinate expression of upd and upd3, the genetic and molecular relationship of upd, upd3 and os was dissected. The os alleles, oso, oss and os1, are a group of classical alleles which display outstretched wings, small eyes, or both, respectively. The genetic complementation tests of upd, upd3 and os showed that both upd and upd3 failed to complement os while upd complemented upd3, suggesting functions of both upd and upd3 are affected in os alleles. Consistent with the genetic tests, the expression of upd and upd3 in eye discs is lost in os allele. Molecularly, putative enhancer regions are deleted at the 5’ end of upd3 in os alleles. Hence, a transcriptional co-regulation model of upd and upd3 is proposed in which upd and upd3 share a common cis-regulatory region, lesions of which cause the os phenotype.

Our lab recently described a role for JAK-STAT signaling in the maintenance of quiescence during the murine hair cycle. Research into signaling pathways and cytokines/growth factors involved in the mammalian hair cycle has not focused extensively on the JAK-STAT pathway. In this thesis, I investigated the upstream effector(s) and downstream mechanisms of JAK-STAT signaling in the HFSC during telogen, using a variety of methods, including murine conditional mutants of the JAK-STAT pathway, pharmacological and immunological techniques. The mechanism through which OSM exerts this effect is via JAK-STAT5 signaling downstream of the OSM receptor, which is antagonized by pharmacological JAK inhibition. Conditional epidermal ablation of OSMR or STAT5 during early- and mid-telogen (P42 – P60) shortens the telogen phase significantly, and inhibition of macrophages by way of neutralizing antibodies, small molecule inhibitors, and genetic ablation (with Csf1r-CreER::R26-iDTR mice) during telogen also promotes hair growth. Single-cell RNA sequencing of dermal immune cells across murine telogen identified a distinct subset of TREM2+ macrophages that are enriched for OSM, and gene-set analysis suggests these “trichophages” are similar to the microglia of the central nervous system. I show that this distinct subset of TREM2+ macrophages predominate during early- and mid-telogen, where they produce Oncostatin M (OSM), which is sufficient to maintain quiescence of hair follicle stem cells (HFSCs). Proliferation of HFSCs and hair growth is associated with depletion of this subset of TREM2+ macrophages. Interestingly, macrophage markers and OSM were found to be upregulated in the balding scalp of males with androgenetic alopecia, suggesting that this mechanism is physiologically relevant in the control of human hair cycling.

Identifying the molecular interactions of pathogens in different mosquito species is critical for understanding how mosquitoes transmit diseases. In this study, the role of the Jak-STAT immune signalling pathway in two different mosquito species, (Aedes aegypti L.) and (Culex quinquefasciatus L.) was assessed. Using in silico analysis tools, cell culture, and molecular techniques, changes in gene expression were assessed during lipopolysaccharide (LPS) challenge and West Nile virus (WNV) infection in the two species. It was found that activation of the Jak-STAT pathway occurred more quickly in Ae. aegypti cells compared to Cx. quinquefasciatus cells during LPS exposure. During WNV infections, no significant differences were observed, although preliminary evidence suggests that differential activation of the Jak-STAT pathway may exist between the two species. This research extends our understanding of the mosquito immune system while demonstrating the critical importance of vector-virus interactions across different mosquito species.

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Identifying the molecular interactions of pathogens in different mosquito species is critical for understanding how mosquitoes transmit diseases. In this study, the role of the Jak-STAT immune signalling pathway in two different mosquito species, (Aedes aegypti L.) and (Culex quinquefasciatus L.) was assessed. Using in silico analysis tools, cell culture, and molecular techniques, changes in gene expression were assessed during lipopolysaccharide (LPS) challenge and West Nile virus (WNV) infection in the two species. It was found that activation of the Jak-STAT pathway occurred more quickly in Ae. aegypti cells compared to Cx. quinquefasciatus cells during LPS exposure. During WNV infections, no significant differences were observed, although preliminary evidence suggests that differential activation of the Jak-STAT pathway may exist between the two species. This research extends our understanding of the mosquito immune system while demonstrating the critical importance of vector-virus interactions across different mosquito species.

Made available in DSpace on 2015-07-22T20:49:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-05-26 / A via de sinalização Janus Kinase/Transdutores de Sinal e Ativadores de Transcrição (JAK/STAT) é uma das mais importantes vias de sinalização para transdução do sinal usadas pelas citocinas. Entretanto, o papel da via JAK/STAT na lesão de isquemia reperfusão (IR) hepática ainda não foi explorado. Este estudo foca no papel da proteína-quinase JAK2, que está acima de STAT-1 na cascata de sinalização JAK/STAT, e o seu papel no mecanismo da lesão de IR hepática. Isquemia quente parcial (lobos esquerdo e médio) foi induzida no fígado de camundongos C57BL/6 por 90 minutos, seguidos por 6 horas de reperfusão. Os animais foram tratados com o inibidor específico de JAK-2 (Tyrphostin AG490; 40 mg/kg, i.p) ou veículo (DMSO), 60 minutos antes do início da isquemia. O bloqueio de JAK2 resultou em significante redução da lesão hepática e da apoptose de hepatócitos. A análise imunohistoquímica revelou uma importante redução no infiltrado de macrófagos e de polimorfonucleares neutrófilos no tecido hepático dos animais que receberam AG490 anteriormente ao insulto isquêmico, comparado aos animais controle (DMSO). A expressão de citocinas pro-inflamatórias (TNF-α, IL-6, IL-1β) e das quimiocinas (CXCL-10 and CXCL-2) também estava reduzida no grupo tratado com AG490, comparado ao grupo controle. A análise da presença de células apoptóticas, através da técnica de TUNEL no tecido hepático dos camundongos, revelou menor número de hepatócitos em processo de apoptose no grupo tratado com AG490 consistente com o achado de western-blot que revelou menor expressão da proteína caspase-3 clivada em paralelo com o aumento da expressão da proteína anti-apoptótica Bcl-XL, no grupo que recebeu AG490. Para confirmar nossos achados in vivo nós testamos AG490 (75 mM) em culturas de macrófagos derivados da medula óssea (BMM) e em cultura de células da linhagem de hepatoma- CRL1830, ambas estimuladas com LPS (10 ng/ml). Em culturas de macrófagos derivados da medula óssea, AG490 reduziu a expressão gênica das citocinas e quimiocinas pró-inflamatórias (IL-6, IL-12p40, IL-1β, CXCL-10 e iNOS) que encontravam-se elevadas apos estimulação com LPS. Em cultura de células da linhagem de hepatoma- CRL1830, AG490 reduziu a expressão de caspase-3 clivada. Além do que, o bloqueio de JAK2 inibiu a fosforilação de STAT1 e STAT3. Pela primeira vez demonstramos que a sinalização por JAK2 é essencial na fisiopatologia da lesão de IR hepática, já que seu bloqueio seletivo protegue o fígado contra inflamação e apoptose. / The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling is one of the major pathways for cytokine signal transduction. However, the role of the JAK/STAT pathway in liver I/R is not clear. This study focuses on JAK2, which functions upstream of STAT-1 in JAK/STAT, and its role in the mechanism of liver IRI. Partial warm ischemia was produced in the hepatic lobes of C57BL/6 mice for 90min, followed by 6h of reperfusion. Mice were treated with JAK-2 inhibitor (Tyrphostin AG490; 40 mg/kg, i.p) or veicle, 60min prior to ischemic insult. JAK2 blockade resulted in significant reduction of hepatocyte apoptosis and liver injury. Macrophage and neutrophil infiltration, as assessed by immunohistochemistry, was markedly decreased in AG490-treated livers, compared with controls. The expression of proinflammatory cytokines (TNF-α, IL-6, IL-1β) and chemokines (CXCL-10 and CXCL-2) was also significantly reduced in AG490-treated group, compared with controls. AG490-treated livers showed less TUNEL positive cells and reduced cleaved caspase-3 protein expression in parallel with increased Bcl-XL expression. We employed AG490 (75 mM) in primary bone marrow-derived macrophage (BMM) and hepatoma cell (CRL1830) cultures, both stimulated with LPS (10 ng/ml). In BMM cultures, AG490 depressed otherwise LPSinduced pro-inflammatory gene expression programs (IL-6, IL-12b, IL-1β, CXCL-10 and iNOS). In hepatoma cells, AG490 reduced cleaved-caspase-3 expression. Moreover, JAK2 blockade inhibited STAT1 and STAT3 phosphorylation. This is the first report, which documents that JAK2 signaling is essential in the pathophysiology of liver IRI, as its selective blockage ameliorated the disease process and protected livers from inflammation and apoptosis. / TEDE / BV UNIFESP: Teses e dissertações / 103 p.

Tumour heterogeneity is a key hurdle for the effective treatment of cancer using oncolytic viruses (OVs). A better understanding of the pathways involved in delineating tumour cell resistance and hypersensitivity to OVs is critical in order to guide the development of new therapeutic strategies to enhance OVs. In this thesis, I performed a comparative genetic and epigenetic study of the murine OV-resistant colon cancer cell line CT26.WT and its hypersensitive subclone CT26.lacZ. This study led to the identification of retroviral insertion sites in AKAP7 and TP53RK genes, that are potentially involved in conveying sensitivity to infection by OVs and the dysregulation of the interferon antiviral response in the CT26.lacZ cell line. Gene overexpression and gene silencing experiments suggest a functional role of these proteins in controlling viral growth. Further investigation of these genes and their relationship to antiviral response pathways is warranted and may lead to novel strategies for improving the therapeutic activity of OVs.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13534号 / 論医博第2274号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 伊藤 貴浩, 教授 江藤 浩之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM