Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves: 138-170). Also available on the Internet.

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<p>Protein tyrosine kinases play an important role in the regulation of various cellular processes such as</p><p>growth, differentiation and survival. GTK, a novel SRC-like cytoplasmic tyrosine kinase, was recently cloned from a mouse insulinoma cell line and the present work was conducted in order to find a biological function of GTK in insulin producing and neuronal cells. It was observed that kinase active GTK-mutants, expressed in RINm5F cells, transferred to the cell nucleus and increased the levels of the cell cycle regulatory protein p27^KIP1, reduced cell growth and stimulated glucagon mRNA expression. Furthermore, wild type GTK induces neurite outgrowth in the rat adrenal pheochromocytoma PC12 cell line, through activation of the RAP1-pathway, suggesting a role of GTK for cell differentiation. Studies using transgenic mice, expressing GTK under the control of the rat insulin 1 promoter, demonstrated a dual role of GTK for β-cell growth: Whereas GTK increases the β-cell mass and causes enhanced β-cell proliferation in response to partial pancreatectomy it also induced β-cell death in response to proinflammatory cytokines and impaired the glucose tolerance in mice treated with the β-cell toxin streptozotocin suggesting a possible role of GTK for β-cell destruction in Type 1 diabetes. We have also observed that GTK-transgenic islets and GTK-expressing RINm5F cells exhibit a reduced insulininduced activation of the insulin receptor substrate (IRS-1 and IRS-2)-pathways, partly due to an increased basal activity of these. GTK was found to associate with and phosphorylate the SH2 domain adapter protein SHB, which could explain many of the GTK-dependent effects both in vitro and in vivo. In summary, the present work suggests that the novel tyrosine kinase GTK is involved in various signal transduction pathways, regulating different cellular responses, such as proliferation, differentiation and survival.</p>

<p>Formation of blood vessels occurs through two processes: vasculogenesis and angiogenesis, which are regulated by various growth factors such as vascular endothelial growth factor, fibroblast growth factor and platelet-derived growth factor. The present study was carried out in order to investigate the roles of the Shb and Cbl proteins in growth factor-mediated signal transduction and blood vessel formation. Shb was found to be involved in NGF-stimulated Rap1 signaling in PC12 cells by forming a complex with CrkII and a 130-135 kDa protein. The Rap1 signaling pathway contributed to NGF-dependent neurite outgrowth. In immortomouse brain endothelial (IBE) cells, Shb increased cell spreading, migration and cytoskeletal rearrangements. Such effects may partly be due to altered Rap1 activation in Shb overexpressing IBE cells. Shb was required for tubular morphogenesis in collagen gels in the presence of FGF-2. In embryoid bodies (EBs) derived from murine embryonic stem cells, Shb up-regulated both VEGFR2 and Tal1 expression at early stages of EB development and thus promoted blood vessel formation both in the absence and in the presence of growth factors. In IBE cells, Cbl positively regulated FGF-2 signaling and increased cell proliferation. Mutation of RING finger alone did not affect blood vessel formation in EBs. However, EBs overexpressing the oncogenic form Cbl 70Z, which had a deletion of the linker region and the first cysteine of the RING finger, exhibited intense CD31 positive sheet-like staining and blood vessel. The results suggested that Cbl had dual roles in endothelial cells: it promoted FGF-2-induced proliferation whereas down-regulated proliferation of endothelial progenitor cells.</p><p>The present work suggests that Shb and Cbl play a crucial role in cell differentiation and blood vessel formation.</p>

The oncogene Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and was initially discovered as the fusion protein NPM (nucleophosmin)-ALK in a subset of Anaplastic Large Cell Lymphomas (ALCL). Since then more fusion proteins have been identified in a variety of cancers. Further, overexpression of ALK due to gene amplification has been observed in many malignancies, amongst others neuroblastoma, a pediatric cancer. Lately, activating point mutations in the kinase domain of ALK have been described in neuroblastoma patients and neuroblastoma cell lines. In contrast, the physiological function of ALK is still unclear, but ALK is suggested to play a role in the normal development and function of the nervous system. By employing cell culture based approaches, including a tetracycline-inducible PC12 cell system and the in vivo D. melanogaster model system, we aimed to analyze the downstream signalling of ALK and its role in neuroblastoma. First, we wished to analyze whether ALK is able to activate the small GTPase Rap1 contributing to differentiation/proliferation processes. Activated ALK recruits a complex of the GEF C3G and CrkL and activates C3G by tyrosine phosphorylation. This activated complex is able to activate Rap1 resulting either in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cells suggesting a potential role in the oncogenesis of neuroblastoma driven by gain-of-function mutant ALK. Next, we could show that seven investigated ALK mutations with a high probability of being oncogenic (G1128A, I1171N, F1174L, F1174S, R1192P, F1245C and R1275Q), are true gain-of-function mutations, respond differently to ALK inhibitors and have different transforming ability. Especially the F1174S mutation correlates with aggressive disease development. However, the assumed active germ line mutation I1250T is in fact a kinase dead mutation and suggested to act as a dominant-negative receptor. Finally, ALK mutations are most frequently observed in MYCN amplified tumours correlating with a poor clinical outcome. Active ALK regulates mainly the initiation of MYCN transcription in human neuroblastoma cell lines. Further, ALK gain-of-function mutants and MYCN synergize in transforming NIH3T3 cells. Overall, somatic mutations appear to be more aggressive than germ line mutations, implying a different impact on neuroblastoma. Further, successful application of ALK inhibitors suggests a promising future for the development of patient-specific treatments for neuroblastoma patients.

Protein tyrosine kinases play an important role in the regulation of various cellular processes such as growth, differentiation and survival. GTK, a novel SRC-like cytoplasmic tyrosine kinase, was recently cloned from a mouse insulinoma cell line and the present work was conducted in order to find a biological function of GTK in insulin producing and neuronal cells. It was observed that kinase active GTK-mutants, expressed in RINm5F cells, transferred to the cell nucleus and increased the levels of the cell cycle regulatory protein p27KIP1, reduced cell growth and stimulated glucagon mRNA expression. Furthermore, wild type GTK induces neurite outgrowth in the rat adrenal pheochromocytoma PC12 cell line, through activation of the RAP1-pathway, suggesting a role of GTK for cell differentiation. Studies using transgenic mice, expressing GTK under the control of the rat insulin 1 promoter, demonstrated a dual role of GTK for β-cell growth: Whereas GTK increases the β-cell mass and causes enhanced β-cell proliferation in response to partial pancreatectomy it also induced β-cell death in response to proinflammatory cytokines and impaired the glucose tolerance in mice treated with the β-cell toxin streptozotocin suggesting a possible role of GTK for β-cell destruction in Type 1 diabetes. We have also observed that GTK-transgenic islets and GTK-expressing RINm5F cells exhibit a reduced insulininduced activation of the insulin receptor substrate (IRS-1 and IRS-2)-pathways, partly due to an increased basal activity of these. GTK was found to associate with and phosphorylate the SH2 domain adapter protein SHB, which could explain many of the GTK-dependent effects both in vitro and in vivo. In summary, the present work suggests that the novel tyrosine kinase GTK is involved in various signal transduction pathways, regulating different cellular responses, such as proliferation, differentiation and survival.

Formation of blood vessels occurs through two processes: vasculogenesis and angiogenesis, which are regulated by various growth factors such as vascular endothelial growth factor, fibroblast growth factor and platelet-derived growth factor. The present study was carried out in order to investigate the roles of the Shb and Cbl proteins in growth factor-mediated signal transduction and blood vessel formation. Shb was found to be involved in NGF-stimulated Rap1 signaling in PC12 cells by forming a complex with CrkII and a 130-135 kDa protein. The Rap1 signaling pathway contributed to NGF-dependent neurite outgrowth. In immortomouse brain endothelial (IBE) cells, Shb increased cell spreading, migration and cytoskeletal rearrangements. Such effects may partly be due to altered Rap1 activation in Shb overexpressing IBE cells. Shb was required for tubular morphogenesis in collagen gels in the presence of FGF-2. In embryoid bodies (EBs) derived from murine embryonic stem cells, Shb up-regulated both VEGFR2 and Tal1 expression at early stages of EB development and thus promoted blood vessel formation both in the absence and in the presence of growth factors. In IBE cells, Cbl positively regulated FGF-2 signaling and increased cell proliferation. Mutation of RING finger alone did not affect blood vessel formation in EBs. However, EBs overexpressing the oncogenic form Cbl 70Z, which had a deletion of the linker region and the first cysteine of the RING finger, exhibited intense CD31 positive sheet-like staining and blood vessel. The results suggested that Cbl had dual roles in endothelial cells: it promoted FGF-2-induced proliferation whereas down-regulated proliferation of endothelial progenitor cells. The present work suggests that Shb and Cbl play a crucial role in cell differentiation and blood vessel formation.

The imidazoline I1 receptor (I1-R) is a novel receptor found primarily in the brain and nervous tissue where it modulates neurotransmission. It is named for its high affinity for compounds with an imidazoline structure such as the anti-hypertensive drugs, clonidine and moxonidine. The imidazoline receptor antisera-selected protein (IRAS) is the putative clone of the I1-R. IRAS has a unique structure, which does not resemble any other receptor protein. IRAS is present throughout the body with highest levels in the brain. There is a growing body of research examining the physiological roles of IRAS as an I1-R, in cell survival, migration and protein trafficking. However, there is little research into its neuronal functions. IRAS interacts with other membrane receptors: the mouse homologue of IRAS reorganises the actin cytoskeleton through interaction with the α5β1 fibronectin receptor. IRAS also binds insulin receptor substrate 4 and enhances insulin-induced extracellular signal-regulated kinase1/2 (ERK1/2) activation. Actin reorganisation and ERK1/2 activation are important for the development of neurites during neuronal differentiation. Therefore, the work described in this thesis aimed to investigate the effects of IRAS on neuronal differentiation. Studies reported in this thesis also aimed to investigate whether IRAS affected ERK1/2 signalling of other receptors involved in neuronal differentiation such as the NGF receptor, TrkA, and lysophospholipid receptors. The above aims were carried out in neuronal model PC12 cells transfected with either IRAS or a vector plasmid. Fluorescence microscopy and Western blotting techniques were used to examine the effect of IRAS on cell morphology and ERK1/2 signalling. The work described in this thesis found that IRAS reorganises the actin cytoskeleton and enhances growth cone development in PC12 cells. This study also shows that IRAS differentially enhances or inhibits NGF-induced PC12 cell differentiation depending on the presence or absence of serum in the media. In full-serum conditions, IRAS enhanced neurite outgrowth and this was accompanied by an increase in ERK1/2 activation. In serum-starved cells, IRAS inhibited neurite outgrowth with similar levels of ERK1/2 activation observed in vector- and IRAS-transfected cells. Finally, studies presented in this thesis found that IRAS enhances lysophosphatidic acid-induced ERK1/2 activation and that IRAS interacting with lysophospholipid receptor agonists present in serum is a potential mechanism by which it enhances NGF-induced ERK1/2 activation in full-serum conditions. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345359 / Thesis (Ph.D.) - University of Adelaide, School of Medical Sciences, 2008

Calcium ions (Ca2+) are involved in almost all neuronal functions, providing the link between electrical signals and cellular activity. This work examines the mechanisms by which a neuron can regulate the movement and sequestration of Ca2+ through specific channels such that this ubiquitous ion can encode specific functions. My initial focus was using intracellular calcium ([Ca2+]i) imaging techniques to study the influence of the inhibition of specific voltage gated calcium channels (VGCC) by ethanol on a depolarization induced rise in [Ca2+]i in neurohypophysial nerve terminals. This research took an unexpected turn when I observed an elevation of [Ca2+]i during perfusion with ethanol containing solutions. Control experiments showed this to be an artifactual result not directly attributable to ethanol. It was necessary to track down the source of this artifact in order to proceed with future ethanol experiments. The source of the artifact turned out to be a contaminant leaching from I.V. drip chambers. Due to potential health implications stemming from the use of these drip chambers in a clinical setting as well as potential artifactual results in the ethanol field where these chambers are commonly used, I choose to investigate this phenomenon more rigorously. The agent responsible for this effect was shown to be di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer that has been shown to be carcinogenic in rats and mice. The extraction of this contaminant from the I.V. drip chamber, as measured by spectrophotometry, was time-dependent, and was markedly accelerated by the presence of ethanol in the solution. DEHP added to saline solution caused a rise in [Ca2+]i similar to that elicited by the contaminant containing solution. The rise in calcium required transmembrane flux through membrane channels. Blood levels of DEHP in clinical settings have been shown to exceed the levels which we found to alter [Ca2+]i. This suggests that acute alterations in intracellular calcium should be considered in addition to long-term effects when determining the safety of phthalate-containing plastics. As part of a collaboration between Steven Treistman and Robert Messing's laboratory at UCSF, I participated in a study of how ethanol regulates N-type calcium channels which are known to be inhibited acutely, and upregulated in the chronic presence of ethanol. Specific mRNA splice variants encoding N-type channels were investigated using ribonuclease protection assays and real-time PCR. Three pairs of N-type specific α-subunit Cav2.2 splice variants were examined, with exposure to ethanol observed to increase expression of one alternative splice form in a linker that lacks six bases encoding the amino acids glutamate and threonine (ΔET). Whole cell electrophysiological recordings that I carried out demonstrated a faster rate of channel activation and a shift in the voltage dependence of activation to more negative potentials after chronic alcohol exposure, consistent with increased expression of ΔET variants. These results demonstrate that chronic ethanol exposure not only increases the abundance of N-type calcium channels, but also increases the expression of a Cav2.2 splice variant with kinetics predicted to support a larger and faster rising intracellular calcium signal. This is the first demonstration that ethanol can up-regulate ion channel function through expression of a specific mRNA splice variant, defining a new mechanism underlying the development of drug addiction. Depolarizing a neuron opens voltage gated Ca2+ channels (VGCC), leading to an influx of Ca2+ ions into the cytoplasm, where Ca2+ sensitive signaling cascades are stimulated. How does the ubiquitous calcium ion selectively modulate a large array of neuronal functions? Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca2+ current and the resulting rise and decay of [Ca2+]i, showing that equal amounts of Ca2+ entering through N-type and L-type voltage gated Ca2+ channels result in significantly different [Ca2+]i temporal profiles. When the contribution of N-type channels was reduced, a faster [Ca2+]i decay was observed. Conversely, when the contribution of L-type channels was reduced, [Ca2+]i decay was slower. Potentiating L-type current or inactivating N-type channels both resulted in a more rapid decay of [Ca2+]i. Channel-specific differences in [Ca2+]i decay rates were abolished by depleting intracellular Ca2+ stores suggesting the involvement of Ca2+-induced Ca2+ release (CICR). I was able to conclude that Ca2+ entering through N-type, but not L-type channels, is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR generates a unique intracellular Ca2+ signal depending on the route of entry, potentially encoding the selective activation of a subset of Ca2+ -sensitive processes within the neuron.

This thesis examines the effects of nerve growth factor (NGF) and lithium on the regulation of neurotensin/neuromedin N (NT/N) gene expression in PC12 pheochromocytoma cells. In PC12 cells, the expression of the rat NT/N gene is strictly dependent on simultaneous exposure to combinations of NGF, glucocorticoids, activators of adenylate cyclase, and lithium. Transient transfection experiments indicated that a consensus AP-1 site located within the NT/N promoter is the principal target of NGF and lithium action. NGF rapidly, but transiently, induces the expression of several AP-1 genes in PC12 cells, suggesting that the effect of NGF on NT/N gene expression results from increased AP-1 activity. These results led to the prediction that the induction of NT/N gene expression should be rapid, transient and dependent on de novoprotein synthesis. These experiments also suggested that the NT/N gene is principally regulated through the initiation of transcription. However, post-transcriptional mechanisms may also be involved. Experiments in this thesis were designed to examine the regulatory mechanisms responsible for increased NT production in PC12 cells when treated with different inducer combinations and whether AP-1 factors could act as mediators in responses to NGF and lithium. Results described in this thesis indicate that the principal mechanism by which NGF and lithium regulate NT biosynthesis is by activating NT/N gene transcription. Comparison of NT/N mRNA, pro NT/N synthetic rates, proNT/N proteins and mature NT levels in induced PC12 cells, demonstrated that NGF and lithium had no effect on the translation of NT/N mRNA and had only a modest effect on post-translational processing. Nuclear run-on assays showed that NT/N transcription is transicntly activated in maximally induced cells. A rapid RNase protection assay was developed to examine both the kinetics of NT/N gene activation and whether activation requires newly synthesized proteins. Quantitation of nuclear NT/N precursor RNA. using a probe spanning the junction between exon onc and intron one, provides a sensitive measure of NT/N gene activity and by several criteria provides an accurate measure of NT/N transcription. When either NGF or lithium was combined with dexamethasone and forskolin, nuclear NT/N precursor RNA transiently accumulated, although each inducer displayed different kinetics, rapid and delayed, respectively. De novo protein synthesis was not required for activating NT/N transcription when NGF was used as the permissive agent, although newly synthesized proteins secm to be needed for subsequent down-regulation. The response to lithium displayed a marked requirement for new protein synthesis, consistent with the involvement of newly synthesized AP-1 factors. RNA blot analysis showed that lithium either alone or in combination with dexamethasone and forskolin induced c-jun and fra-1 gene expression with delayed kinetics, consistent with c-Jun/Fra-1 complexes mediating the effects of lithium on NT/N gene transcription. The pathway identified by lithium does not activate or require protein kinase C. This pathway is also active in neuronally-differentiated PC12 cells suggesting that it could be involved in the regulation of NT/N gcne exprcssion in the intact nervous system. These results and order of addition experiments demonstrate that NGF and lithium activate distinct pathways required for NT/N gene induction.