Resistance of various cancers to conventional therapies including radio- and chemo- therapy is one of the most investigated phenomena in the molecular and clinical oncology. Recurrent disease is characterized by the presence of metastases, which are responsible for 90% of cancer-related mortality. Fractionated ionizing radiation (fIR) combined with surgery or hormone therapy represent the first-choice treatment for medium to high risk localized prostate carcinoma (PCa). In PCa, the failure of radiotherapy (RT) is often caused by radioresistance and further dissemination of escaping (surviving) cells. To investigate the radioresistance-associated phenotype, we exposed four metastasis- derived human PCa cell lines (DU145, PC-3, LNCaP, and 22RV1) to clinically relevant daily fractions of ionizing radiation (fIR; 35 doses of 2 Gy) resulting in generation of two surviving populations: adherent senescent-like cells expressing common senescence-associated markers and non-adherent anoikis-resistant stem cell-like cells with active Notch signaling and expression of stem cell markers CD133, Oct-4, Sox2, and Nanog. While the radioresistant adherent cells were capable to resume proliferation shortly after the end of irradiation, the non- adherent cells started to proliferate only after their reattachment...

Neutrophils form a first line of defence against infections. These short-lived, terminally differentiated cells perform many important functions, including chemotaxis, degranulation, reactive oxygen species (ROS) release and cytokine production. Whilst neutrophils are essential for host immunity, their inappropriate recruitment, activation and/or removal can contribute to excessive inflammation and host damage, as exemplified in autoimmune diseases such as rheumatoid arthritis. It is therefore essential that neutrophil function is tightly regulated. Neutrophils are activated by a range of stimuli, including immune complexes. Neutrophil functions are tightly regulated by intracellular signalling events that are induced by the ligation of cell surface receptors, for example, the binding of immune complexes to Fc receptors. Phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (Erk) are key signalling intermediates that act downstream of many cell surface receptors. They are involved in the regulation of numerous biological processes in the neutrophil. Using pharmacological interventions, I analysed PI3K signalling in immune complex-stimulated human neutrophils and uncovered a previously uncharacterised, noncanonical signalling pathway, PI3K-Cdc42-Pak-Mek-Erk. This represents an unusual situation where Pak acts as the MAP3K downstream of Cdc42 in a PI3K-dependent fashion. By performing a range of functional experiments, I showed that this unconventional signalling pathway promotes apoptosis in human neutrophils by regulating the ratio between anti- and pro-apoptotic members of the Bcl-2 family proteins. No other immune complex-induced, PI3K-dependent neutrophil function tested depended on PI3K-Cdc42-Pak-Mek-Erk signalling. Mouse knock-outs of all components of this signalling pathway have been described. Immune complex-induced apoptosis was also PI3K-dependent in mouse neutrophils, but experiments performed with inhibitors showed that, in contrast to human neutrophils, this was not dependent on PI3K-Cdc42-Pak-Mek-Erk signalling. The myeloid leukaemia cell line, PLB-985 is amenable to knock-down and can be differentiated to become neutrophil-like. These cells are not notably activated by immune complexes, perhaps because they do not express the major Fcγ receptor, CD16. Since retroviral expression of CD16 in PLB985 cells did not improve their response to activation by immune complexes, I was not able to confirm my observations with human neutrophils genetically. Collectively, I showed that a novel, pro-apoptotic signalling pathway operates downstream of Fcγ receptors in the human neutrophil. The fact that this signalling pathway appears to regulate apoptosis specifically suggests uncoupling pro- and anti-inflammatory effects induced by immune complexes might be possible. This may be helpful in the design of improved therapies of autoimmune diseases such as rheumatoid arthritis, in which immune complex-driven neutrophilic inflammation contributes to disease pathogenesis and where neutrophil apoptosis is disturbed.

The goal of this dissertation research is to determine novel regulatory mechanisms of neurotrophin signaling mediated by protein phosphatase 2A (PP2A). PP2A is a ubiquitous Ser/Thr phosphatase that removes phosphates from proteins to switch their activity on or off. The substrate specificity and subcellular localization of PP2A is determined by almost 20 regulatory subunits that associate with a core dimer built of catalytic and scaffold subunits. Since there are more than 48 possible heterotrimers, studying the function of PP2A poses many challenges. Therefore we have devised a strategy, using scaffold subunit knockdown and mutant replacement, to discern the function of specific families of regulatory subunits. With this approach, I have identified specific PP2A holoenzymes that modulate nerve growth factor (NGF) signaling pathways by positively regulating TrkA receptor tyrosine kinase activity. Many studies have shown that NGF is required for the survival and differentiation of sensory and sympathetic neurons. Additionally, NGF is implicated in many neurodegenerative diseases including Alzheimer's disease, Parkinson's disease as well as neuropathic pain. NGF elicits its biological effect through sustained activity of the TrkA receptor and stimulated signaling cascades, including the MAP kinase pathway. Although PP2A has been shown to modulate the mitogen-activated protein (MAP) kinase pathway both positively and negatively at multiple levels, work described herein introduces yet another level of regulation. Specifically, I have shown that PP2A/B' holoenzymes complex with the TrkA neurotrophin receptor to potentiate receptor tyrosine kinase activity, downstream effector kinase activation, neurite outgrowth, and neuronal differentiation. On the other hand, extracellular signal regulated kinase (ERK), a terminal effector in the MAP kinase pathway was shown to phosphorylate a residue in the juxtamembrane region of TrkA and impose feedback inhibition of receptor activity. Collectively, these data suggest a model in which PP2A and ERK oppose each other in the regulation of TrkA receptor activity and downstream signaling cascades that govern neuronal differentiation and maintenance.

Fear and anxiety disorders are potentially crippling conditions that often stem from past experience of trauma and chronic stress. One clear feature of these disorders is the failure to use proximate spatial and contextual information presented in the environment to regulate reflexive physiological threat responses. The central nervous system networks that govern spatial navigation and contextual learning and memory are a series of complex circuitries in which the hippocampus is integrally involved. Deficits in hippocampal function have been linked to severe anterograde and mild retrograde amnesia of semantic and episodic memory, and specific deficits in contextual processing. These deficits manifest as failure to distinguish between the details of contexts that help predict for danger or safety and can thus lead to the overexpression of threat responses that compose the behavioral symptoms of fear disorders. The dentate gyrus (DG) is a subdivision of the hippocampus that serves as the first filter of excitatory flow through the hippocampus. The DG is hypothesized to function in “pattern separation” or the dissociation of similar inputs into dissimilar outputs. Failure in this domain leads to generalization between contexts, a common feature of pathology. Pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1 receptor are associated with multiple behavioral disorders such as post-traumatic stress disorder, schizophrenia, and bipolar disorder. Mutations in the PAC1 receptor gene are associated with hypervigilance, and modified amygdalar and hippocampal activity. These results are mirrored by rodent studies where central PACAP infusion causes anxiety-like behavior, pain hypersensitivity, anorexia, and reinstatement of drug-seeking. PAC1 receptor transcript is found in high abundance in granule cells of the dentate gyrus and potentiation of DG synapses is impaired in PAC1 knockout mice. PACAP is known to have effects of long-duration, such as those in injury repair, growth, and development, but it also can affect ion channel physiology to control neuronal excitability through several parallel intracellular signaling cascades including those dependent on adenylyl cyclase, phospholipase C, and extracellular signal regulated kinase. Accumulated evidence suggests that recruitment of extracellular signal regulated kinase can be through either adenylyl cyclase-, phospholipase C-, or a receptor endocytosis-dependent mechanism. The experiments described in this dissertation address the role of PACAP in the DG in regulating expression of fear behavior, the effects of PACAP on the excitability of DG granule cells, and the signaling pathways and ion channels responsible for these effects. We found that PACAP infused into the DG amplifies expression of fear to a context but does not affect fear acquisition. Electrophysiology studies demonstrate that treating DG neurons with PACAP increases their excitability, and that parallel signaling mechanisms recruit extracellular signal regulated kinase to drive this excitability. Furthermore, these effects on excitability are attenuated by blocking a persistent inward sodium current. This work represents novel regulation of the DG and its impacts on behavior and identifies a current that likely participates in modulating granule cell excitability in multiple domains. In aggregate, this research traces the path from ligand, to receptor and intracellular signaling, to neurophysiology in order to propose a comprehensive description of behavioral regulation by these processes.

This study explored angiotensin IV’s (Ang IV) affects on the signaling pathways involved in the development of insulin resistance in 3T3-L1 adipocytes. Ang IV, working through the AT4 receptor, interferes with insulin signaling through the blockade of the phosphatidylinositol 3-kinases (PI3K)/Akt pathway and through activating mitogen activated protein kinases (MAPK): extracellular signal regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) which are known to impair insulin receptor substrate-1 (IRS-1) signaling. The expression of AT4 receptors was confirmed by reverse transcription polymerase chain reaction. Ang IV’s effects were found by treating adipocytes with combinations of Ang IV, AT4 receptor inhibitor Norleual, and insulin. Cell lysates were resolved by SDS-PAGE electrophoresis and immunoblotted. Ang IV down-regulated the PI3K/Akt pathway. Insulin exerts its effects on adipocytes by activating this pathway, phosphorylating Akt (S473 and T308) residues. Pre-treatment with Ang IV blocked insulin’s effects, reversing Akt activation. Addition of Norleual blocked Ang IV’s inhibitory actions, leading to the phosphorylation of Akt residues. Studies show elevated MAPK levels produced by angiotensin peptides catalyze the phosphorylation of serine residues on IRS-1, impairing insulin signal transduction. Ang IV increased the activation of ERK 1/2 and JNK. This was reversed by pretreatment with Norleual. Ang IV’s effects on IRS-1 residues were found by immunoprecipitation of IRS-1 followed by SDS-PAGE immunoblotting. Ang IV increased the phosphorylation of IRS-1(S307 and S612). Pre-incubation with Norleual attenuated Ang IV’s effects. Ang IV’s stimulation of adipocytes quickly caused the phosphorylation of MAPK corresponding to serine residue phosphorylation on IRS-1, which may implicate Ang IV activation of MAPK in the development of insulin resistance. Ang IV is involved in the down-regulation of the insulin-signaling cascade by inhibiting insulin’s phosphorylation on Akt (S473 and T308). Ang IV increased phosphorylation of ERK 1/2 and JNK, corresponding with increases in serine phosphorylation of IRS-1. Pre-treatment with Norleual inhibited Ang IV’s negative effects on insulin signaling. This study elucidates a new mechanism that may lead to the development of insulin resistance in adipose tissue.

The purpose of this study was to determine whether Endothelin-1 (ET-1) induced cellular responses in bovine corneal endothelial cells (BCECs) involves MAPK pathway by phosphorylating ERK1/2 protein kinase and to find out the phosphorylation patterns of ERK1/2 in confluent and sub-confluent cells. BCECs were isolated from bovine corneas and cultured in medium supplemented with 10% serum. Confluent (contact inhibited) and sub-confluent (actively growing cells) serum starved cells grown in T-75 flasks were treated with 10nM Endothelin-1. The control cells were left untreated. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of phosphorylated ERK1/2 (pp44, pp42) proteins relative to overall ERK1/2 (p44, p42) was determined by western blotting technique. Densitometry analysis of immunoblots revealed differential phosphorylation patterns in confluent and sub-confluent cultures. The pERK1/2 levels were significantly increased at 15 min and 24 hrs after post incubation with ET-1, whereas following the initial rise levels declined to 6hrs of incubation with ET-1 in confluent cultures. In sub-confluent cultures pERK1/2 levels increased gradually to 6hrs of incubation with ET-1, returning to pre-incubation levels at 24hrs. In conclusion, ET-1 treatment was shown to induce phosphorylation of ERK1/2 in BCEC. ET-1 treatment in confluent and sub confluent BCEC exhibited time dependent phosphorylation of ERK1/2. ET-1 treatment affected the phosphorylation pattern distinctively in confluent and sub-confluent BCEC. These observations led to the conclusion that ET-1 induced cellular events in BCEC may involve the MAPK cascade and that these ET-1 induced MAPK cascades may exhibit a negative feedback mechanism, suggested by a distinctive oscillations in pERK 1/2 levels. The contrasting effects of ET-1 in confluent and subconfluent cells may suggest a density dependent phosphatase activity.

LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there is no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In this study, we found increased LIV-1 expression in a progresssive manner in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives prostate cancer EMT in an androgen-refractory human prostate cancer cell (ARCaP) bone metastasis model. LIV-1, when overexpressed in ARCaPE cells (derivative cells of ARCaP with epithelial phenotype), promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells, eliciting constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. Further investigation of the HB-EGF promoter revealed that both Stat3 and AP-1 controlled HB-EGF promoter activity. Ectopic LIV-1 overexpression induced AP-1 and Stat3 activation. Blockade of both Stat3 and AP-1 by specific inhibitors or dominant negative expression vectors diminished the HB-EGF promoter activity induced by LIV-1 overexpression. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promotes EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.

Zebrafish, Danio rerio, have become an important model for developmental studies and have several advantages over other model systems. These advantages include (1) the easy accessibility of zebrafish embryos for direct observation of their development and (2) their suitability for systematic mutagenesis studies for the identification of genes regulating the development of various tissues and organs, including the skeletal system. Recently, it has been reported that glutamate receptors are expressed in many types of bone cells and regulate bone physiological functions. In the present study, we have examined the effects of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist¡Xdextromethorphan¡Xon the development of the axial skeleton in zebrafish embryos by using calcein stain. Our results revealed that dextromethorphan significantly attenuates the formation of the axial skeleton and that it is inhibited on pretreatment with glutamate. Moreover, immunohistochemical analysis revealed protein level expression of the NMDA subunit NR1 in the axial region of zebrafish. Our results also indicate that attenuation of NMDA receptor activity-induced change in the axial skeleton may be related to heat-shock protein and extracellular signal-regulated kinase (ERK) signalings. In conclusion, we suggest that the NMDA receptor plays an important role in the development of the axial skeleton. However, further studies are required on the cellular mechanisms of glutamate regulated bone formation.

ROGDI, a novel gene, locates on human¡¦s chromosome 16p13.3. According to Gene Ontology Annotation database, ROGDI is related to hemopoiesis and positive regulation of cell proliferation. In order to investigate the function of this novel gene in pulmonary fibrosis, fibrotic models in vivo and in vitro were created. Mice which received single intra-tracheal bleomycin injection were sacrificed on various intervals. Rogdi and other pro-fibrotic mediators, including CCL2 and TGF-£]1, were up-regulated in the early phase(< 10 days). On contrary, the anti-fibrotic mediators IL-10, IFN-£^ and heme oxygenase(HO)-1 were up-regulated in the late phase(> 10 days). The precursor microRNA 21 (miR-21) was up-regulated as the fibrotic severity increased. The human embryonic fibroblasts(WI-38 cells) showed fibrogenic phenotype and up-regulation of precursor miR-21 and ROGDI after bleomycin treatment. Human embryonic fibroblasts transfected by coding sequence of ROGDI showed up-regulated precursor miR-21 and £\-SMA compared to those transfected by empty vectors after bleomycin treatment. Two signaling molecules related to positive regulation of cell proliferation, Akt and Erk, showed over-expressed after ROGDI transfection and bleomycin treatment compared to those with empty vector transfection. Our results imply that ROGDI is up-regulated in pulmonary fibrosis and turns fibroblasts into fibrogenic phenotype through positive regulation of miR-21. The increase of precursor, but not primary miR-21, after ROGDI transfection and bleomycin treatment indicates that ROGDI may regulate the TGF-£] signaling pathway in human embryonic fibroblasts. Our results support that ROGDI is a novel gene for pulmonary fibrosis and warrants for further investigation. £[

名古屋大学博士学位論文 学位の種類:博士(リハビリテーション療法学) (課程) 学位授与年月日　平成22年3月25日