Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca2+ concentration ([Ca2+]o) in the body. The secretion of this hormone is suppressed at high [Ca2+]o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca2+ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca2+]o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca2+]o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca2+]o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca2+]o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca2+-independent. The magnitude of neomycin-induced release of Ca2+ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca2+ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca2+]o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca2+ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.

In the lung, epithelial cell migration plays a key role in both physiological and pathophysiological conditions. When the respiratory epithelium is injured, the epithelial lining in the respiratory system can be seriously damaged. Spreading and migrating of the surviving cells neighboring a wound are essential for airway epithelial repair. When the repair process is affected, aberrant remodeling may occur, which is important in the pathogenesis of lung diseases. However, in comparison with other cellular and molecular functions in the respiratory system, our understanding on lung epithelial cell migration and invasion is limited. To gain insight into the molecular mechanisms that govern these cellular processes, I asked whether normal (non-cancerous) human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls cell migration and invasion. I found that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased the invasiveness of these epithelial cells. I further demonstrated that PDBu-induced podosome formation was mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes, whereas atypical PKCζ played a dominant role in the proteolytic activity of podosomes through recruitment of MMP-9 to podosomes, and MMP-9 secretion and activiation. I also found that that PDBu can activate PI3K/Akt/Src and ERK1/2 and JNK but not p38. PI3K, Akt and Src were critical for podosome formation, whereas ERK1/2 and JNK mediated the proteolytic activity of podosomes via MMP-9 recruitment, gene expression, release and activation without affecting podosome assembly. Podosomes are important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration. My thesis work unveils the molecular mechanisms that regulate podosomal formation and proteolytic function in normal human bronchial epithelial cells. These novel findings may enhance our understanding of cell migration and invasion in lung development and repair. Similar mechanisms may be also applicable to other cell types in distinct organs.

Contractility of vascular smooth muscles (VSMs) in resistance arteries determines systemic blood pressure and blood supplies to local tissues, in which ATP sensitive K+ (KATP) channels play a role. The KATP channels that couple metabolic state to cellular activity are activated by multiple hormonal vasodilators and inhibited by vasoconstrictors. To understand the molecular mechanisms for the channel regulation by vasodilators, we studied the effects of β-adrenergic receptors on Kir6.1/SUR2B in HEK cells. Stimulation of β-adrenergic receptors activated the channels, which relied on the GS-protein, adenylyl cyclase, cAMP and PKA system. Using mutational analysis, we scanned all the putative PKA sites on Kir6.1 and SUR2B subunits and identified two residues (Ser1351 and Ser1387) in SUR2B critical for channel activation. In vitro phosphorylation experiments confirmed that Ser1387 but not Ser1351 was phosphorylated in isolated SUR2B peptides. Molecular modeling and molecular dynamics simulations reveal that phosphorylation at Ser1387 causes interdomain movements in SUR2B subunit. Blockage of the movements by engineering a disulfide bond across NBD2 and TMD1 eliminated the PKA-dependent channel activation. We also studied the molecular basis for the inhibition of vascular KATP channels by PKC. In the HEK expression system, we found that the Kir6.1/SUR2B channel but not the Kir6.2/SUR2B was drastically inhibited by PKC stimulation. We constructed Kir6.1/Kir6.2 chimeras and identified two critical protein domains for the Kir6.1 channel inhibition by PKC. The distal C-terminus was the direct target of PKC where multiple phosphorylation sites were identified. These phosphorylation sites were located in a short sequence with stereotypical sequence repeats. Mutation of any decreased the effects of PKC. Joint mutation of all of them prevented the channel inhibition by PKC. The proximal N-terminus is also involved in PKC effects without phosphorylation sites, suggesting it may play a role in channel gating. Thus, this thesis provides experimental evidence for the vascular KATP channel modulation by PKA and PKC. Phosphorylation of the Kir6.1 and SUR2B subunits by PKC and PKA produce inhibition and activation of the vascular KATP channel, respectively, which appears to be one of the molecular bases contributing to vascular tone regulation by both vasoconstricting and vasodilating hormones and neurotransmitters.

In the lung, epithelial cell migration plays a key role in both physiological and pathophysiological conditions. When the respiratory epithelium is injured, the epithelial lining in the respiratory system can be seriously damaged. Spreading and migrating of the surviving cells neighboring a wound are essential for airway epithelial repair. When the repair process is affected, aberrant remodeling may occur, which is important in the pathogenesis of lung diseases. However, in comparison with other cellular and molecular functions in the respiratory system, our understanding on lung epithelial cell migration and invasion is limited. To gain insight into the molecular mechanisms that govern these cellular processes, I asked whether normal (non-cancerous) human airway epithelial cells can form podosomes, a cellular structure discovered from cancer and mesenchymal cells that controls cell migration and invasion. I found that phorbol-12, 13-dibutyrate (PDBu), a protein kinase C (PKC) activator, induced podosome formation in primary normal human bronchial epithelial cells, and in normal human airway epithelial BEAS2B cells. PDBu-induced podosomes were capable of degrading fibronectin-gelatin-sucrose matrix. PDBu also increased the invasiveness of these epithelial cells. I further demonstrated that PDBu-induced podosome formation was mainly mediated through redistribution of conventional PKCs, especially PKCα, from the cytosol to the podosomes, whereas atypical PKCζ played a dominant role in the proteolytic activity of podosomes through recruitment of MMP-9 to podosomes, and MMP-9 secretion and activiation. I also found that that PDBu can activate PI3K/Akt/Src and ERK1/2 and JNK but not p38. PI3K, Akt and Src were critical for podosome formation, whereas ERK1/2 and JNK mediated the proteolytic activity of podosomes via MMP-9 recruitment, gene expression, release and activation without affecting podosome assembly. Podosomes are important for epithelial cell migration and invasion, thus contributing to respiratory epithelial repair and regeneration. My thesis work unveils the molecular mechanisms that regulate podosomal formation and proteolytic function in normal human bronchial epithelial cells. These novel findings may enhance our understanding of cell migration and invasion in lung development and repair. Similar mechanisms may be also applicable to other cell types in distinct organs.

Certain membrane lipids are involved in intracellular signalling processes, among them phosphoinositides and diacylglycerol (DAG). They mediate a variety of functions, including the effects of nutrients and neurohormonal stimuli on insulin secretion from pancreatic β-cells. To ensure specificity of the signal, their concentrations are maintained under tight spatial and temporal control. Here, live-cell imaging techniques were employed to investigate spatio-temporal aspects of lipid signalling in the plasma membrane of insulin-secreting β-cells. The concentration of phosphatidylinositol 4-phosphate [PtdIns(4)P] increased after stimulation with glucose or Gq protein-coupled receptor agonists. The glucose effect was Ca2+-dependent, whereas the receptor response was mediated by isoforms of novel protein kinase C (PKC). The increases in PtdIns(4)P were paralleled by lowerings of the phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] concentration. This relationship was not caused by conversion of PtdIns(4,5)P2 to PtdIns(4)P but rather reflected independent regulation of the two lipids. Stimulation of β-cells with glucose or a high K+ concentration induced pronounced, repetitive increases in plasma-membrane DAG concentration, which were locally restricted and lasted only for a few seconds. This pattern was caused by exocytotic release of ATP, which feedback-activates purinergic P2Y1-receptors and stimulates local phospholipase C-mediated DAG generation. Despite their short durations the DAG spikes triggered local activation of PKC. Novel PKCs were recruited to the plasma membrane both after glucose and muscarinic receptor stimulation. While the glucose-induced translocation was synchronized with DAG spiking, muscarinic stimulation induced sustained elevation of the DAG concentration and stable membrane association of the kinase. Also conventional PKCs translocated to the membrane after glucose and receptor stimulation. The glucose-induced response was complex with sustained membrane association mirroring the cytoplasmic Ca2+ concentration, and superimposed brief recurring translocations caused by DAG. Interruption of the purinergic feedback loop underlying DAG spiking suppressed insulin secretion. Since the DAG spikes reflected exocytosis events, a single-cell secretion assay was established, which allowed continuous recording of secretion dynamics from many cells in parallel over extended periods of time. With this approach it was possible to demonstrate that insulin exerts negative feedback on its own release via a phosphatidylinositol 3,4,5-trisphosphate-dependent mechanism.

The most active form of Vitamin D, 1alpha,25(OH)2D3, modulates cells via receptor mediated mechanisms. While studies have elucidated the pathway via the classical nuclear Vitamin D Receptor (VDR), little is known about the membrane-associated Vitamin D Receptor (ERp60). Caveolae and its characteristic protein Caveolin-1 have been involved in many signaling pathways due to its specific structure and physical configuration. Other studies have shown that many components of the Vitamin D pathway have been found in caveolae. This study hypothesizes that caveolae and Caveolin-1 are important for the effects of 1,25 Vitamin D signaling via ERp60. Research up to date have shown that in rat and mouse growth zone chondrocytes, cells deprived of intact caveolae either through disruption through beta-Cyclodextrin or genetic knockout do not exhibit the characteristic responses to Vitamin D through ERp60 when compared to chondrocytes with functional caveolae. Studies using immunofluorescence co-localization and caveolae fractionation have shown that ERp60 is localized in the caveolae domains. Cellular fractionation was also performed to examine the localization of the ERp60 receptor in lipid rafts and caveolae. Histology and transmission electron microscopy were also used to examine the physiological importance of caveolae and Caveolin-1 in growth plate morphology and cellular characteristics.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 98-113).

Until recently, the main mechanism of argininosuccinate synthase (AS) regulation was described to exist mainly at the level of transcription. Transcriptional regulation of AS has been shown to be coordinate with eNOS in response to shear stress, hypoxia, tumor necrosis factor á (TNF-á), and PPAR ã agonist troglitizone. However, it is now understood that one level of NO regulation is cellular control of arginine availability to eNOS via post-translational modifications of AS such as phosphorylation. The purpose of this investigation was to determine under what conditions AS is phosphorylated at S328, identify the pathway that AS phosphorylation at S328 plays a role, and how phosphorylation affects AS function in endothelial cells. We developed a phospho-specific antibody directed against pS328 AS and assayed for increases or decreases in phosphorylation relative to physiological factors. We found that AS phosphorylation at S328 occurred when endothelial cells were stimulated with physiological factors that stimulate nitric oxide production through calcium-dependent stimulation of eNOS. Furthermore, by utilizing kinase inhibitors and kinase knockdown experiments, we showed that phosphorylation at S328 significantly decreased when PKCá was knocked down, suggesting that S328 phosphorylation of AS is involved in PKCá signaling. In addition, by confocal microscopy, immunoprecipitation, and membrane fractionation, we showed that phosphorylation at S328 of AS promotes its co-localization with eNOS in the perinuclear region. These findings describe a novel pathway involving AS regulation of nitric oxide production, and may serve as a novel drug target in the restoration of vascular nitric oxide homeostasis.

Short-term synaptic plasticity results from use-dependent activity, lasts on the timescale of milliseconds to minutes, and is thought to underlie working memory and neuronal information processing. Here, we focus on two forms of short-term plasticity: 1) post-tetanic potentiation (PTP), which is induced by high-frequency stimulation, and 2) presynaptic ionotropic receptor-activated synaptic enhancement, which can be produced by the activation of presynaptic glycine receptors. Potentiation of evoked and spontaneous responses is thought to arise from elevations in presynaptic residual Ca2+, which activates one or more molecular targets to increase neurotransmitter release. However, the Ca2+ sensor protein has not yet been identified. The overall goal of this work is to elucidate the Ca2+-dependent mechanisms of short-term plasticity.

Acute lung injury (ALI) is a major cause of mortality after lung transplantation. Recent studies indicate protein kinase C delta (PKCδ) could be an effective target to treat ALI. We have developed a gold nanoparticle (GNP)-peptide hybrid that can inhibit PKCδ signaling. PKCδ inhibitor peptide (PKCi) and 95P2P4 stabilizing peptides were conjugated onto GNP. Physicochemical properties of the nanoformulations were examined. A lung transplant-simulated cell culture model was used to evaluate therapeutic efficacy in vitro. A pulmonary ischemia-reperfusion (IR) model was used to test therapeutic efficacy in vivo. GNP-Peptide hybrids showed good stability with high cellular uptake. GNP-PKCi formulations demonstrated anti-inflammatory and anti-apoptotic effects in vitro. When administered to rats under IR stress, GNP-PKCi formulation improved blood oxygenation, reduced pulmonary edema and histological lung injury. In conclusion, we have successfully formulated a clinically-applicable nanoparticle with therapeutic potential to ameliorate lung injury and inflammation. Our formulation strategy could be used to deliver other peptide-based drugs.