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Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

The c-Jun NH2-terminal kinase (JNK) group of kinases include ten members that are created by alternative splicing of transcripts derived from Jnk1, Jnk2 and Jnk3 genes. The JNK1 and JNK2 protein kinases are ubiquitously expressed while JNK3 is expressed in a limited number of tissues. The JNK signaling pathway is implicated in multiple physiological processes including cell transformation. There is growing evidence that JNK signaling is involved in oncogenesis. Nevertheless, the role that JNK plays in malignant transformation is still unclear. The aim of this thesis is to examine the role of JNK in malignant transformation. For this purpose, I used the Bcr/Abl oncogene as a transforming agent. Bcr/Abl is a leukemogenic oncogene that is created by reciprocal translocation between chromosome 9 and 22. The translocation breakpoint is variable and several different Bcr/Abl isoforms have been identified such as Bcr/AblP185 and Bcr/AblP210, whose expression is associated with different types of leukemia. Bcr/Abl activates the JNK signaling pathway in hematopoietic cells and increases AP-1 transcription activity. Furthermore, dominant negative approaches demonstrate that inhibition of c-Jun or JNK prevents Bcr/ Abl-induced cell transformation in vitro. These data implicate the JNK signaling pathway in Bcr/Abl transformation although the role that JNK might have in this process is unclear. Thus, I examined the importance of JNK signaling in Bcr/Abl-induced lymphoid or myeloid transformation. For this purpose I compared Bcr/AblP185- and Bcr/AblP210- induced transformation of wild-type and JNK1-deficient cells using three approaches: in vitro, in vivo and ex vivo. The results obtained with the in vitro approach suggest that both Bcr/AblP185 and Bcr/AblP210 require JNK activity to induce lymphoid transformation. While JNK1-deficiency inhibits Bcr/AblP210 oncogenic potential in lymphoid cells both in vitro and in vivo, pharmacological inhibition of JNK activity (JNK1 and/or JNK2) blocked Bcr/AblP185 induced malignant proliferation in vitro. The differential requirement for JNK observed in the two Bcr/Abl isoforms can be ascribed to the presence in Bcr/AblP210 of the Dbl domain which can activate the JNK pathway in vitro. In the case of Bcr/AblP210, JNK1 is critical for the survival of the ex vivo derived transformed lymphoblasts upon growth factor removal. This result correlates with the fact that mice reconstituted with Bcr/AblP210 transformed Jnk1-l- bone marrow showed normal malignant lymphoid expansion in the bone marrow yet they had reduced numbers of lymphoblast in the bloodstream and lacked peripheral organ infiltration. Thus JNK1 is essential for the survival of the transformed lymphoblast outside the bone marrow microenvironment in Bcr/AblP210induced lymphoid leukemia. Interestingly, while JNK1 is essential for lymphoid transformation, it is dispensable for the proliferation of transformed myeloblasts. Taken together these results indicate that the JNK signaling pathway plays an essential role in the survival of Bcr/AblP210 lymphoblasts and that JNK-deficiency decreases the leukomogenic potential of Bcr/AblP210 in vivo. Thus, cell survival mediated by JNK may contribute to the pathogenesis of proliferative diseases.

The insulin/IGF-1 signaling pathway plays a pivotal role in life span regulation in diverse organisms. In Caenorhabditis elegans, a PI 3-kinase signaling cascade downstream of DAF-2, an ortholog of the mammalian insulin and insulin-like growth factor-1 (IGF-1) receptor, negatively regulates DAF-16/forkhead transcription factor. DAF-16 then regulates a wide variety of genes involved in longevity, stress response, metabolism and development. DAF-16 also receives signals from other pathways regulating life span and development. However, the precise mechanism by which DAF-16 directs multiple functions is poorly understood. First, in Chapter II, we demonstrate that JNK is a novel positive regulator of DAF-16 in both life span regulation and stress resistance. Our genetic analysis suggests that the JNK pathway acts in parallel with the insulin-like signaling pathway to regulate life span and both pathways converge onto DAF-16. We also show that JNK-1 directly interacts with and phosphorylates DAF-16. Moreover, in response to heat stress, JNK-1 promotes the translocation of DAF-16 into thc nucleus. Our findings define a novel interaction between the stress response pathway (JNK) and the master regulator of life span (DAF-16), and provide a mechanism by which JNK regulates longevity and stress resistance. Next, in Chapter III, we focus on the downstream targets of DAF-16. Here, we used a modified chromatin immunoprecipitation (ChIP) method to identify direct target promoters of DAF-16. We cloned 103 target sequences containing consensus DAF-16 binding sites and randomly selected 33 targets for further analysis. The expression of majority of these genes is regulated in a DAF-16-dependent manner. Moreover, inactivation of more than 50% of these genes significantly altered DAF-16-dependent functions such as longevity, fat storage and dauer diapause. Our results show that the ChIP-based cloning strategy leads to greater enrichment of DAF-16 target genes, compared to previous studies using DNA micro array or bioinformatics. We also demonstrate that DAF-16 is recruited to multiple promoters to coordinate regulation of its downstream target genes. In summary, we identified the JNK signaling pathway as a novel input into DAF-16 to adapt animals to the environmental stresses. We also revealed a large number of novel outputs of DAF-16. Taken together, these studies provide insight into the complex regulation by DAF-16 to control diverse biological functions and eventually broaden our understanding of aging.

p38 mitogen-activated protein (MAP) kinases represent a subgroup of MAP kinases that respond to environmental stress and inflammatory cytokines. p38 MAPK is activated by two upstream kinases, MKK3 and MKK6, by dual phosphorylation on threonine and tyrosine in conserved kinase subdomain VII. Until recently the relative roles of MKK3 and MKK6 have remained unclear. I have undertaken two strategies in an effort to understand the importance of MKK3 as a p38 MAPK activator. First, I cloned and characterized the murine mkk3 gene and determined the structure of the 5'-terminus. Comparison of the murine and human mkk3 genes revealed that the mouse gene encodes a single MKK3 isoform, MKK3b, and the human gene encodes two isoforms, MKK3a and MKK3b. Comparison of the mouse and human mkk3 genes suggests that expression of MKK3a and MKK3b is regulated from different promotors. Analysis of the mkk3 promoter demonstrates that muscle specific expression of murine MKK3b is controlled, in part, by the transcription factors MEF2 and MyoD. Second, I have utilized a gene targeting strategy to disrupt the murine mkk3 gene and to examine the effect on p38 MAPK signaling. I found that there is a p38-specific signaling defect in MKK3 deficient primary mouse embryo fibroblasts (MEF) which correlates with deficits in interleukin (IL)-1 and IL-6 production in response to tumor necrosis factor-α (TNFα) stimulation. In addition there is a defect in TNFα mediated expression of TNFα and macrophage inflammatory proteins (MIP) 1α, MIP1β and MIP2. p38 MAPK-specific signaling defects were also observed in lipopolysaccharide (LPS) stimulated mkk3 (-/-) macrophages. Additionally, mkk3 (-/-) macrophages exhibit defects in LPS and CD40-ligand (CD40L) stimulated IL-12 biosynthesis. Similar data were obtained from CD40L-stimulated mkk3 (-/-) dendritic cells. I also observe that interferon (Ifn)-γ production is diminished during T-helper-1 (TH1) differentiation of CD4+ T-cells derived from mkk3 (-/-) mice. Taken together these data demonstrate a crucial role for p38 MAPK activation by MKK3 in response to the inflammatory cytokine, TNFα and during a TH1 inflammatory response.

INTRODUÇÃO: O melanoma primário da cavidade oral é uma neoplasia agressiva, rara e originada a partir da proliferação de melanócitos malignos da mucosa. Ele representa aproximadamente de 0,2 a 8% de todos os melanomas. Estudos recentes apontam algumas vias moleculares tem sido encontradas por estarem envolvidas na patogenia dos melanomas. Dentre essas vias destaca-se a via proliferativa da MAPK (mitogen activated protein kinase), esta cascata de sinalização está envolvida no controle do crescimento celular, proliferação e migração, e tem sido relacionada com um papel importante no desenvolvimento e progressão do melanoma cutâneo. OBJETIVOS: Analisar a expressão proteica e mutação pontual dos componentes da via MAPK e correlacionar com os dados clínicos-histológicos. MATERIAL E MÉTODOS: Através da imunoistoquímica avaliar a expressão proteica dos anticorpos RAS; BRAF; MEK1; MEK2; ERK1 e ERK2 em 35 casos de melanomas orais organizados em matriz (TMA: Tissue Microarray) e através de pirosequenciamento avaliar a mutação pontual dos genes BRAF; NRAS; KRAS em 14 casos de melanomas orais. RESULTADOS: Idade dos pacientes entre 9 e 91 anos, sem predileção por sexo, 75% caucasianos, 71,42% acometeram o palato, 80% com aspecto histológico grau III. A análise da expressão proteica foi: RAS (28,57%); BRAF (82,85%); MEK1 (0%); MEK2 (51,43%); ERK1 (20%)e ERK2 (74,28%). Na análise molecular observamos mutações para BRAF (9/14 casos) e NRAS (2/14 casos). CONCLUSÃO: Todos os aspectos da via MAPK necessita de outras elucidações em melanomas de áreas foto-protegidas e melanomas de mucosa e comparando diferentes populações. Entretanto, os resultados deste presente estudo apontam importante alterações na cascata RAS-RAF-MEK-ERK e estes são indicadores de prognóstico ruim em melanomas primários da mucosa oral, independente da exposição solar / BACKGROUND: Primary melanoma of the oral cavity is an aggressive and rare neoplasm and originated from the proliferation of malignant melanocytes of the mucosa. It represents approximately 0.2 to 8% of all melanomas. Recent studies indicate some molecular pathways have been found to be involved in the pathogenesis of melanomas. Among these means there is a proliferative MAPK pathway (\"mitogen activated protein kinase\"), this signaling pathway is involved in controlling cell growth, proliferation and migration, and it has been associated with a role in the development and progression of melanoma skin. OBJECTIVES: To analyze protein expression and mutation of components of the MAPK pathway and to correlate with the clinical, histological data. MATERIALS AND METHODS: Using immunohistochemistry to evaluate the protein expression of RAS, BRAF, MEK1, MEK2, ERK1 and ERK2 antibodies in 35 cases of oral melanomas organized array (TMA: Tissue Microarray) and using pyrosequencing to assess the mutation of the BRAF, NRAS, KRAS in 14 cases of oral melanomas. RESULTS: Age of patients between 9 and 91 years, regardless of gender, 75% Caucasian, 71.42% in palate, 80% with histologic grade III. Analysis of protein expression was: RAS (28.57%); BRAF (82.85%); MEK1 (0%), MEK2 (51.43%); ERK1 (20%) and ERK2 (74.28%). Molecular analysis we found BRAF mutations (9/14 cases) and NRAS (2/14 cases). CONCLUSION: All aspects of the MAPK pathway requires further elucidation in melanomas of photo-protected areas and mucosal melanomas and comparing different populations. However, the results of this study indicate important changes in the cascade RAS-RAF-MEK-ERK and these are indicators of poor prognosis in primary melanomas of the oral mucosa, regardless of sun exposure

With an increased popularity for systems-based approaches in biology, a wide spectrum of techniques has been applied to the simulation and analysis of biochemical systems which involves uncertainty and stochasticity. It is particularly concerned with modelling and analysis of metabolic pathways, regulatory and signal transduction networks for understanding intra-cellular pathway behaviour. Typically, parameter estimation in ordinary differential equations(ODEs) models is used for this purpose when there is large number of molecules involved in the reaction system. However this approach is correct when the system is large enough to be deterministic in nature. But there are uncertainty involved in the system and the processes are stochastic in nature due to smaller population molecules participating in the pathway reactions. In this thesis the common theme is the study of uncertainties in the chemical kinetics of biochemical reaction systems associated with various intra-cellular pathways and channels. The study is at the mesoscale of the system, i.e., we study systems that do not have too few molecules disallowing any higher scale system level approximation nor too many where a non-stochastic (mesoscale) system approximation will be valid. In our first study we estimate the parameters in the mitogen activated protein kinase (MAPK) signal transduction pathway involved in the departure from the normal Epithelial Growth Factor(EGF) dose-dependency in prostate cancer cells. A model-based pathway analysis is performed. The pathway is mathematically modelled with 28 rate equations yielding those many ordinary differential equations(ODE) with kinetic rate constants that have been reported to take random values in the existing literature. This has led to us treating the ODE model of the pathways kinetics as a random differential equations(RDE) system in which the parameters are random variables. The most likely set of values of the kinetic rate constants obtained from fitting the RDE model into the experimental data is then used in a direct transcription based dynamic optimization method for computing the changes needed in these kinetic rate constant values for the restoration of the normal EGF dose response. It identifies the parameters, i.e., the kinetic rate constants in the RDE model, that are the most sensitive to the change in the EGF dose response behaviour in the PC3 prostate cancer cells. Biochemical pathways involving chemical kinetics equations in terms of low concen-trations of the model variables can be represented as chemical Langevin equations(CLE) as there is stochasticity involved in the processes. Most CLE systems come with the implicit constraint that the concentration state cannot be negative at any time over the sample path. Due to the inherent stiffness(especially in diffusion coefficient) of the CLE system, it has been difficult for numerical schemes to meet this positivity constraint during numerical simulations. Most available methods resort to heuristics by dropping selective noise terms from the original CLE inconsistent with the mesoscale physics involved in forming the CLE. Other methods take very small time steps thus making the simulation inefficient. In our second study we preserve positivity by using a physically consistent numerical scheme which is a modified form of fully stochastic α method for stiff stochastic differential equation. Ion channels are fundamental molecules in the nervous system that catalyse the flux of ions across the cell membrane. Single ion channel flux activity is comparable to the catalytic activity of single enzyme molecules. Saturating concentrations of substrate induce dynamic disorder in the kinetic rate processes of single enzyme molecules and consequently, develop correlative memory of the previous history of activities. Conversely, binding of substrate ion is known to alter the catalytic turnover of single ion channels. Here, we investigated the possible existence of dynamic disorder and molecular memory in single human TREK1 channel due to binding of substrate/agonist using the excised inside-out patch-clamp technique. Our results suggest that single hTREK1 channel behaves as a typical Michaelis-Menten enzyme molecule with a single high-affinity binding site for substrate K+ ion but with uncertainty in reaction rates.

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-κB, facilitated nuclear translocation of NF-κB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-κB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-κB translocation, which are the characteristics of pulmonary endothelial cell activation.

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京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12969号 / 論医博第2102号 / 新制||医||1012(附属図書館) / 32407 / 新制||医||1012 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山田 泰広, 教授 福田 和彦, 教授 伊達 洋至 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM