Identification of Genes Involved in the C. elegans VAB-1 Eph Receptor Tyrosine Kinase Signaling Pathway

The generation of a functional nervous system requires that neuronal cells and axons navigate precisely to their appropriate targets. The Eph Receptor Tyrosine Kinases (RTKs) and their ephrin ligands have emerged as one of the important guidance cues for neuronal and axon navigation. However, the molecular mechanisms of how Eph RTKs regulate these processes are still incomplete. The purpose of this work was to contribute to the understanding of how Eph receptors regulate axon guidance by identifying and characterizing components of the Caenorhabditis elegans Eph RTK (VAB-1) signaling pathway. To achieve this objective I utilized a hyper active form of the VAB-1 Eph RTK (MYR-VAB-1) that caused penetrant axon guidance defects in the PLM mechanosensory neurons, and screened for suppressors of the MYR-VAB-1 phenotype. Through a candidate gene approach, I identified the adaptor NCK-1 as a downstream effector of VAB-1. Molecular and genetic analysis revealed that the nck-1 gene encodes for two isoforms (NCK-1A and NCK-1B) that share similar expression patterns in parts of the nervous system, but also have independent expression patterns in other tissues. Genetic rescue experiments showed that both NCK-1 isoforms can function in axon guidance, but each isoform also has specific functions. In vitro binding assays showed that NCK-1 binds to VAB-1 in a kinase dependent manner. In addition to NCK-1, WSP-1/N-WASP was also identified as an effector of VAB-1 signaling. Phenotypic analysis showed that nck-1 and wsp-1 mutants had PLM axon over extension defects similar to vab-1 animals. Furthermore, VAB-1, NCK-1 and WSP-1 formed a complex in vitro. Intriguingly, protein binding assays showed that NCK-1 can also bind to the actin regulator UNC-34/Ena, but genetic experiments suggest that unc-34 is an inhibitor of nck-1 function. Through various genetic and biochemical experiments, I provide evidence that VAB-1 can disrupt the NCK-1/UNC-34 complex, and negatively regulate UNC-34. Taken together, my work provides a model of how VAB-1 RTK signaling can inhibit axon extension. I propose that activated VAB-1 can prevent axon extension by inhibiting growth cone filopodia formation. This is accomplished by inhibiting UNC-34/Ena activity, and simultaneously activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex.

The type I antifreeze protein gene family in Pleuronectidae

Antifreeze proteins (AFPs) protect marine teleosts from freezing in icy seawater by binding to nascent ice crystals and preventing their growth. It has been suggested that the gene dosage for AFPs in fish reflects the degree of exposure to harsh winter climates. The starry flounder, _Platichthys stellatus_, has been chosen to examine this relationship because it inhabits a range of the Pacific coast from California to the Arctic. This flatfish is presumed to produce type I AFP, which is an alanine-rich, amphipathic alpha-helix. Genomic DNA from four starry flounder was Southern blotted and probed with a cDNA of a winter flounder liver AFP. The hybridization signal was consistent with a gene family of approximately 40 copies. Blots of DNA from other starry flounder indicate that California fish have far fewer gene copies whereas Alaska fish have far more. This analysis is complicated by the fact that there are three different type I AFP isoforms. The first is expressed in the liver and secreted into circulation, the second is a larger hyperactive dimer also thought to be expressed in the liver, and the third is expressed in peripheral tissues. To evaluate the contribution of these latter two isoforms to the overall gene signal on Southern blots, hybridization probes for the three isoforms were isolated from starry flounder DNA by genomic cloning. Two clones revealed linkage of genes for different isoforms, and this was confirmed by genomic Southern blotting, where hybridization patterns indicated that the majority of genes were present in tandem repeats. The sequence and diversity of all three isoforms was sampled in the starry flounder genome by PCR. All coding sequences derived for the skin and liver isoforms were consistent with the proposed structure-function relationships for this AFP, where the flat hydrophobic side of the helix is conserved for ice binding. There was greater sequence diversity in the skin and hyperactive isoforms than in the liver isoform, suggesting that the latter evolved recently from one of the other two. The genomic PCR primers are currently being used to sample isoform diversity in related right-eyed flounders to test this hypothesis.

Role of hypoxia and hypoxia-inducible factor-1 in tumour immune escape

Previous studies revealed that, upon exposure to hypoxia, tumour cells acquire resistance to the cytolytic activity of IL-2-activated lymphocytes. The MHC class I chain-related (MIC) molecules – comprised of MICA and MICB – are ligands for the activating NKG2D receptor on Natural Killer (NK) and CD8+ T cells. MIC-NKG2D interactions lead to the activation of NK and CD8+ T cells and the subsequent lysis of the tumour cells. The study also showed that the mechanism of the hypoxia-mediated immune escape involves the shedding of MIC, specifically MICA, from the tumour cell surface. The objective of the present study was to determine whether the shedding of MICA requires the expression of hypoxia inducible factor-1 (HIF-1), a transcription factor that regulates cellular adaptations to hypoxia. Exposure to hypoxia (0.5% O2 vs. 20% O2) led to the shedding of MIC from the surface of MDA-MB-231 human breast cancer cells and DU-145 human prostate cancer cells as determined by flow cytometry. Knockdown of HIF-1α mRNA using siRNA technology resulted in inhibition of HIF-1α accumulation under hypoxic conditions as determined by Western blot analysis. Parallel study revealed that knockdown of HIF-1α also blocked the shedding of MICA from the surface of MDA-MB-231 cells exposed to hypoxia. These results indicate that HIF-1 is required for the hypoxia-mediated shedding of MICA and, consequently, that HIF-1 may play an important role in tumour immune escape. Ongoing studies aim to determine the HIF-1 target genes involved in the shedding of MICA under hypoxia.

THE ROLE OF LIPOPROTEIN(a)/APOLIPOPROTEIN(a) IN ENDOTHELIAL DYSFUNCTION: MECHANISTIC STUDIES IN VASCULAR ENDOTHELIUM

Multiple lines of evidence suggest that elevated plasma lipoprotein(a) (Lp(a)) concentrations are a significant risk factor for the development of a number of vascular diseases including coronary heart disease and stroke. Lp(a) consists of a low-density lipoprotein (LDL)-like moiety and an unique glycoprotein, apolipoprotein(a) (apo(a)), that is covalently attached to the apolipoproteinB-100 (apoB-100) component of LDL by a single disulfide bond. Many studies have suggested a role for Lp(a) in the process of endothelial dysfunction. Indeed, Lp(a) has been shown to increase both the expression of adhesion molecules on endothelial cells (EC), as well as monocyte and leukocyte chemotactic activity in these cells. We have previously demonstrated that Lp(a), through its apo(a) moiety, increases actomyosin-driven EC contraction which, as a consequence, increases EC permeability. In this thesis, we have demonstrated a role for the strong lysine-binding site in the kringle IV type 10 domain of apo(a) in increasing EC permeability, which occurs through a Rho/Rho kinase-dependent pathway. We have further validated these findings using mouse mesenteric arteries in a pressure myograph system. We also have dissected another major signaling pathway initiated by apo(a) that involves in a disruption of adherens junctions in EC. In this pathway, apo(a)/Lp(a) activates the PI3K/Akt/GSK3β-dependent pathway to facilitate nuclear translocation of beta-catenin. In the nucleus beta-catenin induced the expression of cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) from the EC. Finally, we have presented data to suggest a novel inflammatory role for apo(a) in which it induces the activation of nuclear factor-kappaB through promotion of the dissociation of IkappaB from the inactive cytoplasmic complex; this allows the nuclear translocation of NFkappaB with attendant effects on the transcription of pro-inflammatory genes. Taken together, our findings may facilitate the development of new drug targets for mitigating the harmful effects of Lp(a) on vascular EC which corresponds to an early step in the process of atherogenesis.