Classically, the epithelial lining of the urinary bladder, also called the urothelium, has been thought of as a passive barrier against toxins present in urine. However, recent studies are beginning to emerge that demonstrate an active role for the urothelium in the sensory functions of the bladder. For example, the urothelium expresses a number of the same receptors as sensory nerves and can respond to and release transmitters. One such transmitter, acetylcholine, has been shown to be released from the urothelium in response to physical stimuli, and is thought to act back on the urothelium in an autocrine/paracrine manner to effect urothelial signaling. This study was undertaken to determine if the urothelium expresses the proper receptors to respond to acetylcholine, specifically nicotinic acetylcholine receptors, and if these receptors play a role in influencing bladder physiology. Our research indicates that the urothelium expresses the proper nicotinic receptor subunits to form two classes of receptor: 1) α3 heteromeric receptors and 2) α7 homomeric receptors. Both of these classes of urothelial receptor are functional and can alter bladder reflexes in the anesthetized rat. Specifically, α7 receptors mediate an inhibitory pathway as measured by a bladder cystometrogram, while α3 receptors mediate an excitatory pathway. Finally, we examined intracellular and extracellular pathways that may mediate these physiological effects in vivo. These experiments suggest that nicotinic receptors in the urothelium mediate their effects through intracellular calcium signaling, resulting in the modulation of the release of the excitatory transmitter ATP. Specifically, our research indicates that α3 stimulation can potentiate the release of ATP from urothelial cells, while α7 stimulation inhibits it. This effect may be due to the fact that each receptor subtype modulates [Ca+2]i through distinct pathways: α3 receptors through influx of extracellular Ca+2 and α7 receptors through release from intracellular stores. Additionally, our research indicates that α7 receptors can inhibit signaling through α3 receptors, indicating another possible mechanism for the inhibitory effects α7 receptors exhibit in vivo. This research, which is the first to indicate an interaction between two types of nicotinic receptor, suggests that urothelial nicotinic receptors could play a significant role in bladder physiology and may represent a viable target for treatments into bladder pathology.

Caveolin-1 (Cav-1) is a membrane associated scaffolding protein that regulates a myriad of signaling molecules. It has been implicated as both a tumor suppressor and promoter. Here, we examine the proteins link to senescence and cancer, and identify a novel pathway through which Cav-1 mediates stress induced premature senescence (SIPS) through p53 activation. Oxidative stress triggers p38MAPK , which activates the transcription factor Sp1. Sp1 binds to two GC-rich regions in the caveolin-1 promoter up-regulating the protein. Cav-1 binds to p53s negative regulator, MDM2, sequestering the E3 ligase to allow p53 to become active. p53 activates its downstream targets, such as p21WAF/CIP1, which initiates SIPS. This pathway is dysfunctional in many cancers that have a downregulated Cav-1 gene. The effects of oxidative stress in Cav-1 null backgrounds were examined. Breast cancer cells that do not express Cav-1 cannot undergo oxidatively induced SIPS. However, upon re-expression of Cav-1, the SIPS phenotype is restored. Utilization of Cav-1 knockout mouse embryonic fibroblasts show that without Cav-1 to sequester MDM2, allowing for the upregulation of p53 leading to SIPS, cells continued to proliferate. These results distinguish Cav-1 as a molecular senescence switch, because in its absence oxidative SIPS does not occur, but in its presence it does. This effect is also not specific to a particular cell type; data supports Cav-1 as a molecular switch in epithelial and fibroblast cell lines. Finally, senescence is known to have antagonistic pleiotropic effects on an organism. That is, cell senescence is beneficial for younger organisms, as it prevents the proliferation of mutated genomes through growth arrest. However, an accumulation of senescent cells can lead to aging and become detrimental. Cav-1s role in the antagonistic pleiotropic effects of senescent fibroblasts on neoplastic epithelial cells is also explored. Data shows that senescence of fibroblasts depends upon Cav-1 sequestering MDM2, which activates p53 and induces SIPS. These fibroblasts can secrete factors that make it advantageous for NIH 3T3 RasG12V transformed fibroblasts and MDA-MB-231 breast cancer epithelial cells to proliferate in vitro and in vivo. Hence, we propose that the Cav-1 gene functions with antagonistic pleiotropy.

The hallmark of chronic myelogenous leukemia (CML) is a chromosomal translocation between the c-abl gene (chromosome 9) and the bcr gene (chromosome 22). This event gives rise to BcrAbl, a chimeric protein with constitutive tyrosine kinase activity that drives the pathogenesis of the disease. Imatinib, a Bcr-Abl kinase inhibitor is the frontline therapy in CML. Although imatinib is very effective in the chronic phase of CML, patients in advanced stages develop resistance. An increased understanding of the signaling pathways implicated in CML pathogenesis and imatinib resistance is critical to the development of improved therapies. Previous work in our laboratory found that A-419259, a broad-spectrum Src family kinase (SFK) inhibitor induces growth arrest and apoptosis in CML cells, suggesting that SFKs are required for Bcr-Abl transformation of myeloid progenitors. Additionally, Hck couples BcrAbl to Stat5 activation in myeloid cells, which may contribute to survival. Furthermore, studies on samples from some imatinib-resistant patients found increased expression and activity of Hck and Lyn. In this dissertation, using two chemical genetic methods, I addressed the contribution of Hck to Bcr-Abl signaling and imatinib resistance. To explore the individual contribution of Hck to Bcr-Abl signaling, I developed an A419259-resistant mutant of Hck (Hck-T338M). Expression of Hck-T338M fully protected K562 CML cells from A-419259-induced apoptosis, an effect that correlated with sustained Stat5 activation. In addition, the Hck-T338M partially protected CML cells against the growth inhibition induced by A-419259. These studies suggest that Hck plays a non-redundant role as a key downstream survival partner for Bcr-Abl. I also tested whether Hck overexpression was sufficient to induce imatinib resistance in CML cells. For this study, I developed a mutant of Hck (Hck-T338A) that is uniquely sensitive to NaPP1, an analog of the generic SFK inhibitor pyrrazolo-pyrimidine 1. Overexpression of Hck or Hck-T338A in K562 cells induced resistance to imatinib-dependent apoptosis and growth arrest. Furthermore, NaPP1 reversed imatinib resistance in K562-Hck-T338A cells, suggesting that Hck-induced imatinib resistance requires Hck kinase activity. Taken together, my work validates Hck as a target for the development of apoptosis-inducing drugs and that are likely to be effective in imatinib-resistant patients.

Structural coupling between the cytoplasmic (CP), transmembrane (TM) and extracellular (EC) domains of G protein coupled receptors (GPCRs) is crucial for their functioning in signal transfer from the extracellular to the intracellular side of the membrane. The focus of this thesis was to test the hypothesis that ligands can bind in each of the three domains. Depending on the location of the endogenous ligand binding site, the other two sites would become allosteric ligand binding sites. To test this hypothesis, we investigated the binding of accessory ligands to each of the three domains, CP, TM and EC. The major contributions of this thesis are as follows: I. The anthocyanin Cyanidin-3-glucoside (C3G) and the chlorophyll-derivative chlorin e6 (Ce6), were shown to physically interact with rhodopsin. These studies demonstrated the presence of a novel CP allosteric ligand binding site in rhodopsin. Biophysical evidence indicated differential effects of binding of these ligands on rhodopsin function, structure and dynamics. II. The allosteric TM ligand binding pocket in metabotropic glutamate receptors (mGluRs) was shown to be analogous in structure and function to the orthosteric TM retinal ligand binding pocket in rhodopsin. Docking of known allosteric modulators to structural models of mGluRs based on rhodopsin conformations was used to predict allosteric modulatory effects. Structural comparison of the mGluR and rhodopsin binding pockets revealed high overlap and preliminary evidence was obtained showing that an mGluR ligand can bind to rhodopsin. III. Evidence for the existence of an EC ligand binding domain was presented. Rhodopsin was shown to bind the extracellular chemokine ligand, CXCL11, an event which interfered with both rhodopsin and chemokine functions. IV. As part of the above efforts, it became necessary to develop and improve NMR spectroscopic methodology to study ligand binding of membrane proteins such as GPCRs. Thus, 1H and 19F based NMR methods to screen for novel ligands that bind to GPCRs were developed and applied to rhodopsin. Collectively, the studies presented in this thesis enhance the understanding of allosteric modulation of GPCRs in general, and of the molecular mechanism of rhodopsin and mGluR activation in the presence of allosteric ligands in particular. The results could help in the identification of new ligands to allosterically modulate receptor structures and in turn their functions at different binding pockets, thus paving new ways to selectively target this pharmacologically important class of receptors.

The mammalian lung supports the transport and diffusion of inspired and expired gasses that are critical for aerobic life. With every inspiration the lung is exposed to environmental agents including microbes, virus, and environmental pollutants. In the event that injury occurs the epithelium is repaired by an abundant facultative progenitor pool and a sequestered population of adult tissue stem cells. Chronic lung diseases, such as asthma, chronic obstructive pulmonary disease, and bronchopulmonary dysplasia, are characterized by extensive epithelial remodeling resulting in a reduction to the number of non-ciliated bronchiolar Clara cells. Given the established role for Clara cells as abundant facultative progenitors, these data suggest that epithelial repair has been compromised. In addition to affects on the epithelium, these diseases are also accompanied by extensive subepithelial fibroproliferation, mesenchymal remodeling, and elevated extracellular matrix deposition as well as a profound increase to lung inflammation. It has been postulated, but never tested in vivo that mesenchymal remodeling and uncontrolled deposition of extracellular matrix may be a result of impaired airway epithelial reparative capacity. Moreover, the finding that airway epithelial cells are essential for modulation of innate immunity suggests that the enhanced inflammatory response described in chronic lung disease may be a result of attenuated airway epithelial cell function. Therefore, this dissertation tests the hypothesis that airway epithelial reparative capacity moderates extracellular matrix deposition and innate immunity. Through the use of in vivo models of injury, inflammation, and attenuated Clara cell function, this dissertation research work identifies a previously uncharacterized process in which extracellular matrix is dynamically and reversibly regulated during productive epithelial repair and severely disrupted by blocking stem cell mediated repair. In addition, the use of mouse models of decreased Clara cell abundance and secretion demonstrate airway epithelium modulates pulmonary innate immunity through regulation of macrophage behavior and inhibition of pulmonary inflammation. This work defines two phenotypes that are the result of attenuated epithelial repair and supports the paradigm that epithelial reparative capacity may be a principal determinant of lung disease.

Hypoxia represents an important element of the solid tumor microenvironment and contributes to tumorigenesis and resistance to chemo- and radiation therapy. Hypoxia can modulate cell cycle progression although the mechanisms involved remain unclear. The Cdc25A dual specificity phosphatase promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases. Cdc25A is the master regulator of the cell cycle and its disruption induces cell cycle arrest in cancer cells. The recent observation that under hypoxic conditions, levels of Cdc25A protein and mRNA are decreased led to the hypothesis that hypoxia-mediated reduction in Cdc25A may represent a novel mechanism in the hypoxic regulation of the cell cycle. Given the prominent role of Cdc25A in regulating the cell cycle and the proposed changes in Cdc25A protein and mRNA under hypoxic conditions, it was hypothesized that Cdc25A plays an essential role in hypoxia-mediated cell cycle arrest in human tumor cells. The specific aims were to: 1) examine the mechanism of Cdc25A downregulation in response to hypoxia, 2) determine the role HIF-1α in Cdc25A regulation and cell cycle arrest, and 3) determine if Cdc25A downregulation is required for hypoxia-induced cell cycle arrest. Under hypoxic conditions, Cdc25A protein levels were specifically and reversibly suppressed. It was found that Cdc25A mRNA levels are significantly decreased by a p21-dependent mechanism. In addition, suppression of Cdc25A was independent of p53. Loss of Cdc25A protein occurred in the absence of checkpoint activation. Recent evidence has linked the microRNA miR-21 to Cdc25A and hypoxia. It is shown here that miR-21 was required for Cdc25A mRNA suppression in hypoxic colon cancer cells and miR-21 levels were increased under hypoxic conditions. The HIF-1α transcription factor was not required for suppression of Cdc25A but must be present for hypoxia-induced cell cycle arrest. Under hypoxic conditions, cells undergo p21- and miR-21- S-phase cell cycle arrest. This study proposes a novel mechanism of transient regulation of Cdc25A via the p21- and miR-21-dependent regulation of mRNA levels in hypoxic cells leading to cell cycle arrest. This previously unknown mechanism may confer protection from hypoxic conditions, contributing to cell survival and the observed resistance to chemo- and radiation therapy.

Head and neck squamous cell carcinoma (HNSCC) is characterized by the overexpression of the epidermal growth factor receptor. However, molecular targeting strategies against EGFR have not improved the 5-year survival rates of HNSCC patients. EGFR tyrosine kinase inhibitors displayed limited clinical responses in Phase II trials and the FDA-approved monoclonal antibody cetuximab (C225) did not prevent the occurrence of secondary tumors and distant metastases. G-protein-coupled receptor ligands; gastrin-releasing peptide (GRP), prostaglandin E2 (PGE2) and bradykinin (BK) have all been reported to activate EGFR in HNSCC via extracellular release of EGFR ligands TGF-á and AR. To improve the efficacy of EGFR inhibition in HNSCC, we investigated the efficacy of targeting common signaling intermediates involved in GPCR-EGFR crosstalk. We previously reported that GRP mediated release of EGFR ligands via phosphoinositide-dependent kinase 1 (PDK1) dependent phosphorylation of a disintegrin and metalloprotease 17 (ADAM17). We subsequently investigated whether PDK1 mediates EGFR activation downstream of PGE2, BK and LPA pathways and the efficacy of different PDK1 targeting strategies in HNSCC. PGE2, BK and LPA-mediated EGFR phosphorylation was abrogated in PDK1 siRNA-transfected HNSCC cells. PDK1 siRNA also decreased PGE2 and BK-mediated HNSCC growth in vitro. Expression of kinase-dead PDK1 (PDK1M) decreased PGE2 -mediated HNSCC growth. PDK1M HNSCC cells demonstrated reduced proliferation compared to control HNSCC cells. HNSCC cells displayed nanomolar sensitivity to the PDK1 inhibitor OSU-03012 compared to normal mucosal cells. Combined treatment with the EGFR TKIs erlotinib or AG1478, plus OSU-03012 enhanced anti-proliferative effects. We have reported that PGE2 and BK mediated MAPK phosphorylation in the presence of EGFR inhibition, and combined GPCR and EGFR demonstrated additive to synergistic anti-tumor effects. To elucidate the EGFR-independent signaling mediated by GPCRs, we used a forward phase phosphoprotein array to identify potential molecular targets that can potentiate EGFR inhibition. We observed that p70S6K phosphorylation was induced in EGFR siRNA-transfected cells and sustained in cetuximab (C225)-treated cells following PGE2 or BK stimulation. Further investigation showed that p70S6K phosphorylation mediated by EGFR downmodulation was dependent on PDK1 and PKCä expression. Combined targeting of EGFR with cetuximab and p70S6K with the mTOR inhibitor RAD001 decreased GPCR-mediated growth in vitro and in vivo. The results from this study have indicated that targeting the GPCR signaling intermediates PDK1 and p70S6K in conjunction with EGFR may be beneficial therapeutic strategies for the subset of HNSCC patients that respond poorly to cetuximab treatment.

In the United States in 2009, 192,370 women are expected to be diagnosed with invasive breast cancer, and 62,280 with in situ disease. About 70% of these cases are estrogen receptor positive (ER+). There are two isoforms of the ER, α and β, that differ somewhat in structure and action. ERβ expression plays a protective role in breast cancer, and selective targeting of this isoform would have many beneficial effects. Tamoxifen has long been the standard of care for patients with ER+ breast cancer. A major problem with tamoxifen is the development of drug resistance. One of the mechanisms proposed for the development of tamoxifen resistance involves the loss of ERβ expression. The first objective of this study was to screen a library of biphenyl C-cyclopropylalkylamides for their ability to function as ERβ-selective ligands. Two compounds were identified with modest selectivity for ERβ and anti-proliferative effects in breast cancer cells where they inhibited expression of c-Myc. The nuclear matrix (NM), the structural scaffolding of the nucleus, plays a major role in many fundamental processes of the cell. Using the ER+ breast cancer cell line MCF-7 and an antiestrogen resistant derivative, along with subtype selective ER ligands, alterations in the abundance of specific proteins present in the NM were identified using a mass spectrometry (MS)-based relative quantitative methodology. Some of the most interesting proteins with altered abundance are NuMA, serpin H1, hnRNP R, and dynein heavy chain 5. These proteins may represent putative biomarkers to customize treatment. The alterations also provide a mechanistic understanding of tamoxifen resistance. The NM was also investigated by MS in the earliest stage of breast cancer, ductal carcinoma in situ (DCIS), utilizing novel cell lines derived from normal (breast reduction), DCIS, and non-diseased contralateral breast surgical specimens. Two of the interesting proteins found to be altered in DCIS were HSP90 and EEF1D. These studies may provide biomarkers to aid in the diagnosis and treatment of breast cancer. In addition by understanding the mechanism behind the development of breast cancer, prevention becomes a possibility.

Caveolin-3 is a muscle specific scaffolding protein with both structural and signaling roles. Lack of caveolin-3 expression has been implicated in limb-girdle muscular dystrophy, along with distal myopathy and rippling muscle disease. These diseases are characterized by progressive muscle weakness and muscle wasting. Nicotinic acetylcholine receptor (nAChR) clustering and localization are important for efficient nerve to muscle contractile signal transmission. It is hypothesized that muscle weakness could originate through disrupted nAChR clustering, disrupting the efficiency of signaling from the motorneuron to the muscle. While the molecular mechanisms involved in nAChR clustering remain to be fully defined, we hypothesize caveolin-3 is important for nAChR clustering and overall neuromuscular junction function. Caveolin-3 and the nAChR co-localize and associate evidenced by immunofluorescence and immunoprecipitation. These results were replicated in differentiated wildtype myotubes treated with the nAChR clustering agent, neural agrin. In differentiated caveolin-3 null myotubes, agrin treatment yields a 60% reduction in nAChR clusters as compared to agrin treated wildtype myotubes. Agrin induces nAChR clustering, through activation of muscle specific kinase (MuSK) and downstream through Rac-1 activation. In differentiated wildtype myotubes, Rac-1 activation peaks at 1 hour of agrin treatment, while in differentiated caveolin-3 null myotubes there is dramatically reduced Rac-1 activation upon agrin treatment. Immunoprecipitation of MuSK shows that caveolin-3 and MuSK association peaks at 1 hour of agrin treatment in wildtype cells. This corresponds to the peak of MuSK phosphorylation which also occurs at 1 hour. Agrin induced MuSK phosphorylation was decreased more significantly than the overall decrease in MuSK expression in the caveolin-3 null cells as compared to the wildtype results. These results indicate a role for caveolin-3 in efficient nAChR clustering. Electromyography studies in anesthetized mice indicated lengthened latencies of the muscle action potential in the caveolin-3 null mice as compared to wildtype mice. There were also decreased overall electromyography (EMG) amplitude and EMG area under the curve in caveolin-3 null mice. Comparison of contractile strength in wildtype and caveolin-3 null animals indicated tetanic contractions to be less stable in the caveolin-3 null animals, though there was late potentiation in actual contractile strength. Lack of caveolin-3 affects the neuromuscular junction formation and transmission without affecting overall contractile strength. This research opens a novel view, that correct neuromuscular junction formation and neuromuscular transmission is important in the development of muscular dystrophies.

Telomeres help maintain the overall genomic stability of an organism, and telomeric homeostasis is critical to navigating between aging and cancer. Telomeric dysfunction is implicated as a contributing factor in numerous aging-related diseases, such as diabetes, impaired hematopoeisis, and atherosclerosis. Telomeric homeostasis is maintained by a shelterin complex of six proteins and an array of telomere-associated proteins that interact with the central shelterin complex, such as the Werner syndrome helicase/exonuclease protein (WRN) or p53. Telomeres also have non-canonical DNA structures that are critical towards their function, especially G-quadruplex DNA (G4 DNA) and Holliday Junctions. The former are pseudoknots that form on the G-rich 3 single-stranded tail of the telomere and may block telomere replication and lengthening, when the 3 telomeric tail is exposed in the open conformation. We found that the shelterin protein protection of telomeres 1 (POT1) competes with and destabilizes G4 DNA on a physiologically realistic telomeric tail substrate, leading to an equilibrium population of diminished G4 DNA coexisting with POT1. While POT1 is a passive binder of DNA, the destabilizing effect of bound POT1 on pre-existing G4 DNA leads to an emergent, de facto cooperativity in G4 DNA unfolding by POT1. Holliday Junctions (HJ) form when the telomere is in a closed conformation, in which the 3 telomeric tail invades the duplex telomeric DNA, creating a displacement loop (D-loop) and sequestering the end of the chromosome from unwanted DNA damage responses. The D-loop is a homologous recombination intermediate, and we demonstrate that telomere repeat binding factor 2 (TRF2) is necessary to protect HJ DNA from unwanted WRN helicase activity, which has been thought to branch migrate the D-loop into a target for Holliday Junction cleaving enzymes, causing sudden telomere shortening. TRF2 also protects HJ DNA from Holliday Junction cleaving enzymes, and the cleavage protection is due largely to the HJ-binding B-domain on TRF2. In contrast, we found that TRF2-mediated protection against WRN depends on both the B domain and the telomeric-repeat binding Myb domain. We have therefore discovered an overlapping but distinct role for TRF2 in maintaining telomeric stability. Our work has elucidated novel structural and functional data on the modulation of non-canonical DNA structures by shelterin and telomere-associated proteins. These data help us elucidate the mechanisms underlying cellular and animal models for telomere instability and aging.