Wounding disrupts the primary function of an epithelium, which is to provide a barrier to the outside environment. The longer the epithelial defect remains unhealed, the greater the risks of morbidity and mortality from infection, loss of tissue homeostasis, and fibrosis. Normally, epithelial cells restore barrier function by becoming highly motile and migrating to cover the defect, but in many situations cells do not move fast enough to prevent tissue malfunction. This is especially true in the cornea, where even minor wounds can impair vision. Therefore, there is considerable therapeutic interest in identifying signals that induce epithelial migration. Activation of the epidermal growth factor receptor (EGFR) is a key signaling event that promotes cells to move and cover wounds in many epithelia. The broad goal of this dissertation research was to identify mechanisms of wound-induced EGFR activation so that therapies may be developed to improve normal and pathological healing. I hypothesized that mechanisms of EGFR activation may differ with respect to distance from the wound, so I developed wounding models to analyze signaling specifically in cells near to or far from wounds in a human corneal epithelial cell line. I have examined the involvement of extracellular ATP, phospholipase D, Src-family kinases (SFKs), and the focal adhesion kinase Pyk2, all of which are signals that have been hypothesized to be stimulated by environmental cues related to wounding and to activate the EGFR. I have found that the proximal mechanism of EGFR activation is the proteolytic release of membrane-bound ligands, which is regulated by activation of SFKs. After wounding, multiple pathways converge on SFKs to regulate EGFR activation and cell motility. In one pathway, extracellular ATP transactivates the EGFR through phospholipase D2. In a distinct pathway that functions specifically near the wound edge, Pyk2 triggers SFK and EGFR activation. Finally, my data suggest the presence of a third distinct pathway that promotes SFK and EGFR activation in response to a physically unconstrained edge. By delineating signaling pathways that stimulate EGFR activation, I have identified potential therapeutic targets for modulating EGFR signaling and cell motility in wound healing and other pathologies.

Hypoxic pulmonary vasoconstriction (HPV) is a unique physiological response of the lung that acts to optimize gas exchange by diverting blood flow from poorly ventilated regions. The endothelium has been thought to play a mostly modulatory role in this phenomenon through the synthesis of vasoactive agents such as nitric oxide (NO), prostacyclin, and endothelin. Data is provided showing that acute hypoxia induces increases in NO biosynthesis, promoting S-nitrosation of the metal binding protein metallothionein (MT), which resulted in intracellular release of zinc. Hypoxia released zinc induced contraction of pulmonary endothelial cells and contributed to vasoconstriction of small, non-muscularized intra-acinar arteries in isolated perfused mouse lungs (IPL). The relevance of this NO/MT/Zn pathway in HPV was illustrated by pharmacological inhibition of NO synthesis and analysis of the response in MT knockout (MT-/-) mice, both of which resulted in a blunted pressure response to hypoxia in IPL. Signaling pathways were delineated, indicating how changes in intracellular zinc can alter the actin cytoskeleton and promote cellular contraction. It was found that either hypoxia or exogenous zinc resulted in increases in the formation and alignment of actin stress fibers. These changes were mediated through the inhibition of myosin light chain phosphatase (MLCP), which promoted phosphorylation of myosin light chain (MLC) and tension generation. Activation of PKC appeared to play a role in this process, as indicated by activation and translocation of the enzyme in response to both hypoxia and/or increases in labile zinc, and by the blunted contractile response in isolated endothelial cells following pharmacological inhibition of PKC or utilization of a PCKε dominant negative construct. These data suggest that the NO released in response to hypoxia promotes increases in MLC phosphorylation through zinc-dependent pathways, which in turn are responsible for the force induction and cell stability necessary to elicit an active contractile response in pulmonary endothelium.

Ion channels are the molecular units that underlie electrical signaling in cells. Many physiological processes are dependent upon this signaling mechanism, as dysregulation often leads to severe pathophysiological consequences. The intermediate conductance calcium-activated potassium channel (KCa3.1) functions as heteromeric complexes with calmodulin (CaM), which is constitutively bound to the calmodulin-binding domain (CaMBD) of KCa3.1 located in the C-terminus, just distal to the sixth transmembrane domain (S6). This arrangement enables CaM to function as an intracellular Ca2+-sensor, coupling changes in the intracellular Ca2+ concentration to the regulation of channel activity. Understanding how channels gate or transition from the closed to the open conformation is a fundamental question in the field of ion channel biophysics. A chemomechanical gating model was proposed to explain how Ca2+-binding causes the channel to transition from a non-conducting to a conducting configuration. However, this model lacks a specific mechanism explaining how the conformational change in the CaMBD is coupled to the activation gate. Therefore, the goal of this dissertation was to investigate the role of S6 in the activation mechanism of KCa3.1. Specifically, I tested the hypothesis that the non-luminal residues in the C-terminal portion of S6 function as an interacting surface to couple CaM to the activation gate. Biochemical perturbation and site directed mutagenesis targeting predicted non-luminal residues in S6 act to shift the gating equilibrium toward the open state by increasing the apparent Ca2+ affinity and dramatically slowing the deactivation process. Kinetic modeling using a 6-state gating scheme showed these perturbations act to slow the transition between the open state back to the closed state. The modification in the steady-state and kinetic behavior of the channel in combination with the kinetic analysis indicate the shift in gating equilibrium is caused by slowing the closing transition, suggesting the non-luminal surface of S6 is allosterically coupled to the activation gate. Therefore, in addition to being a structural component of the pore; S6 is also a dynamic component of the activation mechanism. Continuing to identify regions of the channel participating in the activation mechanism is critical to understand how Ca2+ binding leads to channel opening.

Phosphatidylinositol (PI) and its phosphorylated derivatives, phosphatidylinositides (PIPs), are versatile cellular regulators participating in myriad events including signal transduction, cytoskeleton organization, protein targeting and many steps of membrane traffic. Different PIPs exhibit non-overlapping distributions on cellular membranes. This feature contributes to organelle identities and is tightly controlled by kinase/phosphatase-mediated PIP synthesis and turnover. Mechanisms regarding compartment-restriction and detailed functions of many PIPs and PI/PIP metabolizing enzymes remain largely unknown. My dissertation focuses on the cellular targeting mechanism of a PIP kinase and the pathogenesis of a disease caused by mutations in a PIP phosphatase. Phosphatidylinositol (4,5)-bisphosphate (PIP2), an apical-surface-enriched PIP in polarized epithelial cells, is primarily synthesized via phosphorylation of phosphatidylinositol 4-phosphate (PI4P) in the presence of type I PI 5-kinases (PI5KIs). Previous studies have suggested that the three isoforms of PI5KI (¦Á, ¦Â, and ¦Ã) exhibit distinct cellular functions. Data from our lab indicate that these three PI5KIs are differentially localized in polarized renal cells. While the majority of ¦Á and ¦Ã isoforms are present on lateral cell surface, the ¦Â isoform strikingly localizes to the apical plasma membrane. Using mutagenesis, immunofluorescence, and confocal microscopy, I have found that the apical surface distribution of PI5KI¦Â is nonsaturable and does not require catalytic activity or the presence of PIP2. These results provide useful information for future studies on PI5KI¦Â-regulated cellular activities. PIP2 turnover can be catalyzed by a variety of enzymes, one of which is OCRL1. OCRL1 is a PI 5-phosphatase that preferentially hydrolyzes PIP2, producing PI4P, and is associated with the trans-Golgi network, endosomes, and clathrin-coated-pits. Genetic defects of OCRL1 cause Lowe syndrome, a disease manifested by congenital cataracts, mental retardation, and renal tubular dysfunction. By examining cultured renal epithelial cells acutely depleted of OCRL1 via RNA interference, I have found that loss of OCRL1 does not interfere with endocytic trafficking of the multiligand receptor megalin, or uptake of megalin ligands. OCRL1 knockdown did appear to disrupt delivery of newly-synthesized lysosomal hydrolases and alter distribution of primary cilia length in renal epithelial cells. These findings suggest that multiple pathways may contribute to development of renal symptoms in Lowe patients.

Clathrin-mediated endocytosis is a selective pathway for the entry of transmembrane proteins into the cell through the generation of a short-lived vesicular intermediate. Cells and tissues depend on this process for obtaining nutrients, modulation of signaling and cell migration. The clathrin-coated structure intermediate is assembled on the plasma membrane from a cohort of 20-30 distinct proteins that aid in cargo selection, scaffolding, membrane bending and scission of the vesicle. Exactly how these complex assemblies are nucleated at the plasma membrane remains unclear although the lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) plays an important role by anchoring many of the endocytic components. The work in this thesis helps to clarify the nucleation phase by describing the molecular details of the interaction between a PtdIns(4,5)P2-generating lipid kinase PIPKIgamma and the heterotetrameric clathrin adaptor AP-2. By engaging a subdomain on the AP-2 beta2 subunit appendage, the kinase is strategically positioned at assembly sites to generate PtdIns(4,5)P2 and drive coat assembly forward. Clathrin binds to the same subdomain on the beta2 appendage but with a higher apparent affinity. I therefore invoke a model in which PtdIns(4,5)P2 production for nucleation is negatively regulated by PIPKIgamma displacement from AP-2 by clathrin at later stages of assembly. I also demonstrate that a cargo-sorting alternate adaptor that binds to the other subsite on the AP-2 beta2 appendage is not subject to displacement by clathrin during clathrin-coated vesicle budding, ensuring non-competitive cargo incorporation into the vesicle. Finally, the PtdIns(4,5)P2-binding EFC domain proteins FCHO1 and FCHO2 have been proposed to act as dedicated nucleators of clathrin-coated structures on the plasma membrane. I demonstrate in multiple cell lines that these proteins are not invariantly required for placement of clathrin-coated assemblies on the plasma membrane despite being early arriving components themselves. FCHO1/2 are involved in the regulation of the size and number of these assemblies in some cellular contexts. My data support the model of PtdIns(4,5)P2 regulated, not protein regulated, nucleation of clathrin-coated structures; however multiple parallel pathways may contribute to initiation of endocytic buds.

Most cells throughout their existence are constantly subjected to enormous amounts of endogenous and exogenous DNA damage. The cellular response to genotoxic stressors ultimately either leads to adaptive processes that mediate cellular repair and allow for continuous cellular function or leads to cell malfunction and death. In some theories of aging this cellular malfunction is due to accumulation of unrepaired DNA damage which over time leads to progressive deterioration of tissue/organ homoeostasis and function resulting in organismal aging. The overall goal of my studies is to understand the responses to DNA damage in corneal endothelial (CE) cells whose pump and barrier functions are essential for corneal transparency and which in vivo display age-related degeneration and accumulation of DNA damage. In three complementary and related studies I have focused on how the CE is affected by genotoxic stress. In the first study I have examined the clinical application of the DNA crosslinking agent mitomycin C during photorefractive keratectomy and documented its effects on the CE such as significant accumulation of DNA lesions and elevated levels of apoptosis. In the second study I have examined the long term consequences resulting from failure to repair endogenous DNA damage in vivo. Using a DNA repair-deficient mouse strain I have observed significant premature age-related dystrophic changes in the CE that only occur in very old mice. This suggests that the CE is sensitive and vulnerable to the effects of accumulating endogenous genotoxic stress and that DNA damage may drive CE aging. In the third study I have examined how CE cell-cell communication mediated by gap junctions is affected by acute genotoxic stress. Given that gap junction intercellular communication is essential for homeostasis and associated with cell proliferation, death and survival, alterations in the gap junction protein connexin-43 may be crucial for CE cell function and viability during genotoxic stress. The key findings of all my studies elucidate the role of genotoxic stress in CE aging and identify novel responses to stresses from DNA damage. Through a greater understanding of the responses to these stressors, efforts to preserve and improve CE viability and function can be achieved.