Browsing by Subject "Cell migration"
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Item Adrenergic regulation of CFTR-dependent anion secretion and cell migration in airway epithelial cells(2014-12) Peitzman, Elizabeth RuthThe overall goal of this thesis was to understand the mechanisms underlying beta-adrenergic receptor (beta-AR) regulation of anion secretion and epithelial cell migration on mucociliary clearance and epithelial repair in human airways. The first chapter of the thesis presents an overview of the literature with the purpose of providing background information of mucociliary clearance, airway epithelial cell restitution and specific inflammatory disease conditions where these processes contribute to the innate defense of the airways. Chapters 2 and 3 describe the methods, results and a discussion of experiments that were performed to address the overall goal of the thesis. The final chapter presents a general discussion of the major findings of these studies in the context of what is presently known about the role of beta-AR regulation of epithelial function in airways. Initial experiments presented in Chapter 2 so looked at the effects of growth conditions on beta-AR localization in a human airway epithelial cell model system (Calu-3 cells). The results from these experiments showed that under air-liquid interface (ALI) conditions basolateral stimulation with 1uM epinephrine produced a significant increase in CFTR dependent anion secretion, whereas cells that were grown under liquid liquid interface (LLI) had a much smaller increase secretion when stimulated with the same concentration of epinephrine. These results indicated that basolateral expression of beta-ARs is increased when cells were grown under ALI. Furthermore Calu-3 cells that were apically stimulated with the beta2-AR selective agonist salbutamol produced an increase in anion secretion similar to what was observed with 8cpt-cAMP, a non-metabolizable analog of cyclic AMP. Additionally, when cells were treated with carvedilol, an inverse agonist acting at beta2-ARs, an initial decrease in basal Isc occurred and carvedilol treatment after stimulation with 8cpt-cAMP inhibited anion secretion. Cells pretreated with nocotazole, an agent that disrupts microtubule assembly, blocked the inhibitory effects of carvedilol on anion secretion, suggesting that endocytosis was necessary in order to observe the inhibitory effects of carvedilol. Finally, western blot analysis of apical membrane proteins showed that when cells were treated with carvedilol there was reduced expression of CFTR in the apical membrane, which was not observed following stimulation with epinephrine.Experiments in chapter 3 were designed to investigate the effects of beta-AR agonists on epithelial cell migration. Stimulation of Normal Human Bronchial Epithelial (NHBE) cells and Calu-3 cells), with a beta2-AR agonist produced a significant increase in time to wound closure compared to untreated control cells. Moreover, agonist stimulated cells were rescued when pretreated with beta-AR antagonists propranolol or ICI-118551. The addition of beta-AR agonists epinephrine or salbutamol to CFTR silenced cells (shCFTR) or cells where CFTR was inhibited with 20uM CFTRinh-172 showed no further decrease in migration rate suggesting that inhibition due to a change in CFTR expression or activity. Furthermore beta-AR agonists reduced lamellipodia protrusion similar to what was observed after CFTR inhibition. Overall these results suggest that treatment of airway epithelial cells with beta-AR agonists causes a decrease in migration rate leading to a significant reduction in the time required for complete wound closure. Additionally these results suggest that stimulating cells with carvedilol causes inhibition of cAMP-stimulated anion secretion by promoting retrieval and internalization of CFTR from the apical membrane. These results signify that the chronic use of beta-AR agonists can lead to increased risk for infection in individuals with obstructive airway disease and the use of bias ligands that promote Gs signaling while blocking beta-arrestin signaling may be the key to effectively treating these patients. These drugs would potentially promote wound repair and mucociliary clearance while limiting the risk of increased exacerbations and infection.Item Fibroblast growth factor Receptor 1-induced osteopontin regulates proinflammatory molecules to mediate cross-talk between breast cancer cells and the surrounding tumor microenvironment(2012-06) Reed, Johanna RaeTumor formation is an extensive process requiring complex interactions that involve both tumor cell-intrinsic pathways and soluble mediators within the microenvironment. Tumor cells exploit the intrinsic functions of many soluble molecules, including cytokines and chemokines and their receptors, to regulate pro-tumorigenic phenotypes that are required for development of the primary tumor. Previous studies demonstrated that activation of inducible FGFR1 (iFGFR1) in mammary epithelial cells resulted in increased proliferation, migration, and invasion in vitro and tumor formation in vivo. Early studies also indicated that iFGFR1 activation stimulated recruitment of macrophages to the epithelium resulting in increased epithelial cell proliferation and angiogenesis. Our current studies further examined this model to identify novel mechanisms that regulate early stage tumorigenesis with a specific emphasis on the soluble mediators that are regulated by iFGFR1 to promote epithelial and stromal cell migration. Results from this study elucidate a novel role for iFGFR1-induced osteopontin in promoting a proinflammatory tumor microenvironment through the regulation of proinflammatory molecules such as IL-1beta; and activation of the COX-2/PGE2 pathway as well as by promoting recruitment of CX3CR1-expressing macrophages through regulation of the chemokine CX3CL1. Defining the role of osteopontin-regulated proinflammatory, secreted molecules in promoting iFGFR1-mediated mammary tumorigenesis is important for understanding how initiating oncogenic events drive tumor growth and progression through the secretion of soluble mediators. Moreover, results from these studies will aid in identifying potential novel molecular targets for therapeutic intervention.Item The Kruppel, the Like, the Factor, the 2, and the Ology.(2010-07) Weinreich, Michael AlexanderThis thesis project began with testing the role of IL-7 and Erk5 in regulation of KLF2 expression and SP maturation. We provide evidence that SP maturation occurs independently of both IL-7 and Erk5. Next we examined the direct effects of KLF2 deficiency in T cells. Here we made the surprising discovery that KLF2 deficiency causes cell extrinsic effects on wild type (WT) thymocytes using mixed bone marrow chimeras. In the third chapter we show that the cell extrinsic effects result in expression of the chemokine receptor CXCR3 and are dependent on IL-4 receptor signaling acting through the transcription factor eomesodermin. The cell extrinsic effects also lead to delayed thymocyte emigration and we investigate the mechanism for this emigration defect in chapter 4. This leads to the novel finding that CXCR3 is necessary for the maintenance of returning memory CD8 T cells in the thymus. In the fifth chapter, we identify a cell intrinsic expansion of PLZF+ T cells as the source of IL-4 in the KLF2 deficient thymus. We also show that the cell extrinsic effects lead to memory phenotype and function on bystander CD8 T cells. We show that this mechanism occurs in other gene deficiency models and in WT BALB/c mice.Item Optimality in the nanomechanics of cell migration and adhesion(2014-09) Bangasser, BenjaminCell migration is key to many biological processes including embryonic development, wound healing, and disease progression, and the mechanical stiffness of a cell's environment exerts a strong, but variable, influence on this migration. Many cells display a stiffness optimum at which migration is maximal, however, these stiffness optima span several orders of magnitude, from ~1-1000 kPa, suggesting that different cell types possess distinct operating parameters. Firstly, we describe how a motor-clutch model of cell traction, which exhibits a maximum in traction force with respect to substrate stiffness, may provide a mechanistic basis for understanding how cells are "tuned" to sense the stiffness of specific microenvironments. We found that the optimal stiffness is generally more sensitive to clutch parameters than to motor parameters, but that single parameter changes are generally only effective over a small range of values. By contrast, dual parameter changes, such as coordinately increasing the numbers of both motors and clutches, offer a larger dynamic range for tuning the optimum. The model exhibits distinct regimes with "frictional slippage" at both low and high substrate stiffness where clutches are inefficiently utilized. Between the two extremes, we find the maximum traction force where clutches are most efficiently utilized, which occurs when the substrate load-and-fail cycle time equals the expected time for all clutches to bind. Secondly, we also present a master equation-based ordinary differential equation (ODE) description of the motor-clutch model, from which we derive an analytical expression for a cell's optimum stiffness. This analytical expression provides insight into the requirements for stiffness sensing by establishing fundamental relationships between the key controlling cell-specific parameters. Both the ODE solution and the analytical expression show good agreement with Monte Carlo motor-clutch output, and reduce computation time by several orders of magnitude, which potentially enables long time scale behaviors (hours-days) to be studied computationally in an efficient manner. The ODE solution and the analytical expression may be incorporated into larger scale models of cellular behavior to bridge the gap from molecular time scales to cellular and tissue time scales. Thirdly, to create a unified theoretical framework for cell migration, we have developed and experimentally tested a whole cell migration simulator based on the motor-clutch model by imposing coupled force balances and mass balances on molecular motors, adhesion molecules ("clutches"), and actin subunits in a compliant microenvironment. The model predicts a stiffness optimum that can be shifted by altering the number of active molecular motors and clutches. This prediction was verified experimentally by comparing cell traction and F-actin retrograde flow for two cell types with differing amounts of active motors and clutches: embryonic chick forebrain neurons (ECFNs; optimum ~1 kPa) and U251 glioma cells (optimum ~1000 kPa). In addition, the model predicted, and experiments confirmed, that the stiffness optimum of U251 glioma cell migration, projected area, aspect ratio, F-actin flow rate, and traction strain energy can be shifted to lower stiffness by simultaneous drug inhibition of myosin II motors and integrin-mediated adhesions. Overall, the motor-clutch cell migration simulator provides a unified theoretical framework with which to predict cell adhesion and migration in defined mechanochemical environments.