Browsing by Subject "Epithelial Cells"
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Item Characterization and Selective Targeting of Aneuploid Human Colonic Epithelial Cells(2012-11-28) Ly, Peter 1986-; Lum, Lawrence; Pearson, Gray W.; Pandita, Tej K.Aneuploidy, an abnormal number of chromosomes, occurs in the vast majority of sporadic colorectal cancer patients. Despite this observation, the cellular advantages conferred by recurring cytogenetic alterations are poorly understood and targeted therapies selective to aneuploid cells are currently non-existent. Here, we provide evidence that serum-free passage of originally diploid, immortalized human colonic epithelial cells give rise to the acquisition of trisomy 7 (1CT+7), an aneuploidy detected in over 40% of colorectal adenomas. Pre-existing 1CT+7 cells within the original population were undetectable through GTG-banding and fluorescent in situ hybridization analysis, suggesting a conversion of diploid cells to an aneuploid state. Compared to their isogenic diploid counterpart, 1CT+7 cells express higher levels of the epidermal growth factor receptor (EGFR, located on chromosome 7p). Treatment with the pharmacological adenosine analog 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) completely halted proliferation of 1CT+7 cells and reduced both metabolic consumption and production in vitro. Unexpectedly, treatment of 1CT+7 cells with AICAR led to a reversible 3.5-fold reduction in EGFR overexpression. AICAR-induced depletion of EGFR protein can be abrogated through inhibition of the proteasome with MG132. AICAR also heavily promoted EGFR ubiquitination in cell-based immunoprecipitation assays, suggesting enhanced degradation of EGFR protein mediated by the proteasome. Moreover, treatment with AICAR reduced EGFR protein levels in a panel of human colorectal cancer cell lines in vitro and in xenograft tumors in vivo. Our data collectively supports the compound AICAR as a novel inhibitor of EGFR protein abundance and as a potential anticancer agent for aneuploidy-driven colorectal cancer. In summary, we have isolated and characterized isogenic human colonic epithelial cells that represent recurrent chromosomal acquisitions in sporadic colorectal cancer and demonstrate how it may be possible to selectively target these cells for therapeutic intervention.Item Characterization of Non-Coding RNAs in Regulating Thymic Epithelial Cell Responses to Pathophysiological Stress(2016-07-26) Hoover, Ashley Renae; Hooper, Lora V.; van Oers, Nicolai S. C.; Cleaver, Ondine; Mendell, Joshua T.The thymus is uniquely sensitive to several forms of stress. Stress initiates a transient involution that can reduce overall thymic volume up to 90%. The thymus is predominantly composed of developing thymocytes and specialized epithelial cells. The type of stress predicates whether the thymocytes or epithelial cells initiate the involutionary response. MicroRNAs (miRs) are small non-coding RNAs ~18-22 nucleotides in length that maintain cellular homeostasis and regulate stress responses. Previous work in the laboratory identified several microRNAs involved in regulating thymocyte responses to stress. Thymocytes have been the main population studied in response to stress. However, it has become increasingly clear that the epithelial cells play a critical role in thymus involution and the subsequent recovery of thymopoiesis. The work presented in this thesis characterizes an epithelial specific miR, miR-205, and its surrounding long noncoding RNA, MIR205.001 in regulating TEC functions. A deficiency of miR-205 specifically in TECs renders these cells more susceptible to stress mediated thymic involution. This is revealed by a significant loss in developing thymocytes, altered migration, delayed recovery of single positive thymocyte selection, and proliferative defects in cortical TECs. Gene expression comparisons revealed miR-205 deficient TECs had reduced levels of the TEC master transcriptional regulator, Foxn1, as well as the expression of multiple chemokines. MiR-205 mimics introduced into miR-205 deficient fetal thymic organ cultures were able to restore the levels of Foxn1 and selected chemokines. This work demonstrates that miR-205 positively regulates Foxn1 and chemokine expression following stress. MiR-205 resides within a putative long noncoding RNA (lncRNA), MIR205.001. TECs deficient in this lncRNA are also sensitive to stress, but do not experience a delay in thymopoiesis nor cortical TEC proliferation defects. This suggests these noncoding RNAs have non-overlapping functions within the TECs. Mice deficient in MIR205.001 also have distinct phenotypes, displaying reduced mendelian ratios indicative of a lethality. The surviving animals display reduced body size, weight, and fat mass. Current experiments are addressing whether this is a metabolic defect or due to changes in feeding behavior.Item Differentiation of Normal and Cystic Fibrosis Human Lung Epithelial Cells in a Decellularized and Reconstituted Mouse Lung(2017-04-14) LaRanger, Ryan; Hsieh, Jenny; Garcia, Christine K.; Pasare, Chandrashekhar; Lu, Christopher Y.; Shay, Jerry W.; Wright, Woodring E.Engineered lung tissue may eventually address the chronic shortage of transplantable lung tissue and permit modeling of lung disease in a controlled ex vivo environment. However, there are presently no sources of primary lung stem cells which can be expanded at sufficient scale to permit engineering multiple lungs from a single donor. I have developed a method for conditionally reprogramming primary human bronchial epithelial cells in culture to extend their functional lifespan, and have used these cells to reconstitute lung epithelium in a decellularized lung matrix. For conditional reprogramming, I cultured primary human bronchial epithelial cells derived from patients with or without cystic fibrosis with a small molecule Rho-associated coiled kinase inhibitor and co-cultured it with irradiated J2 3T3 fibroblasts. I determined the ability of the human bronchial epithelial cells to differentiate after 40 population doublings by culture at an air liquid interface for 35 days as confirmed by transepithelial electrical resistance measurement, histology, Ussing chamber analysis, and immunofluorescence staining of differentiation factors. I also found that this conditional reprogramming method permits cloning of human bronchial epithelial cells, and that these cells can support genetic modification by CRISPR. Next, I developed a method for decellularizing and reconstituting murine lungs in a bioreactor with vascular perfusion and simulated breathing. Lungs reconstituted with the conditionally reprogrammed human bronchial epithelial cells formed both upper and lower airway structures after only 12 days of culture. I confirmed the formation of a bronchial pseudostratified epithelium and alveolar formation in the reconstituted lungs by histology, western blotting, and immunofluorescence staining. To develop an eventual universal donor paradigm for engineered tissue, I developed an in vivo luciferase rejection screen in mice using luciferase expressing life-extended primary skin fibroblasts transplanted intradermally. The methods developed for long-term culture of primary lung epithelial cells permits rapid scale-up of patient derived human bronchial epithelial cells and clonal selection without the need for genetic manipulation; facilitating the study of lung diseases and optimization of organ reconstitution in tissue engineered models.Item Effects of Radiation Exposure on Lung Carcinogenesis(2010-01-12) Delgado, Oliver; Shay, Jerry W.Lung cancer is one of the most prevalent forms of cancer in both men and women with over 1.3 million annual related deaths worldwide. Analysis of several human populations exposed to radiation reveals that the lung is remarkably susceptible to the carcinogenic effects of radiation exposure. The considerable lung surface area and slow rate of epithelial turnover may have causal roles in this vulnerability. This may be due to the increased probability that a progenitor cell of the lung, which is proposed to be the cancer-initiating cell, may acquire multiple carcinogenic alterations from radiation exposure. Currently, the lung is believed to have several facultative progenitor cells, situated throughout the lung epithelium, that are regionally restricted in their regenerative capacity. Normal human bronchial epithelial cells (HBECs), immortalized through the expression of Cdk4 and hTERT, provide a sustainable cell reagent for the evaluation of the radiation effects in vitro. These HBECs retain a novel multipotent capacity in vitro (capable of differentiating into both central and peripheral lung cell types) and thus may represent an unrestricted progenitor of the adult lung that resembles an embryonic progenitor. Studies to determine whether the differentiation state influences radiation exposure effects, such as DNA damage and repair, are ongoing. As cellular responses change upon the acquisition of oncogenic mutations, the effects of fractionated or acute radiation exposure on lung carcinogenesis in vivo were determined utilizing the transgenic LA1 K-ras mouse model of lung cancer compared to wildtype littermates. Radiation-induced carcinogenesis is a major concern not only for cancer patients being treated with therapeutic radiation but also for astronauts on long-term space missions. X-ray radiation did not affect the incidence or progression of lung carcinogenesis in this mouse model of lung cancer. High-energy 56Fe- particle irradiation (a type of radiation present in deep space), however, significantly increased the incidence of invasive carcinoma when administered as a fractionated dose but not as a single acute dose. These results demonstrate that pre-initiated lesions may be more susceptible to malignant transformation upon exposure to radiation. Thus, radiation may have an impact on both lung cancer initiation and progression.Item In Cell Stress Conditions, VEGFR2 Exerts Pronounced Effects on Cell Growth in Dysplastic Barrett's Epithelial Cells(2015-01-26) Belli, Olivia; Zhang, Qiuyang; Souza, Rhonda F.BACKGROUND & AIMS: Vascular endothelial growth factor (VEGF), a potent inducer of angiogenesis, recently has been shown to exert direct pro-proliferative and pro-survival effects on cancer cells through binding to its receptors, VEGFR1 and VEGFR2. In earlier studies, we showed that VEGF/VEGFR2 signaling exerts direct pro-proliferative effects on transformed Barrett's and adenocarcinoma cells in an autocrine fashion, with no significant effects on apoptosis. To explore the potential contribution of VEGFR signaling to cell growth of dysplastic Barrett's cells, we knocked down the VEGFR1 or VEGFR2 and studied the effects on cell morphology, cell number, proliferation, and apoptosis. METHODS: We used 3 high-grade dysplastic Barrett's epithelial cell lines (CP-B, CP-C, and CP-D). VEGFR1 and VEGFR2 were knocked down through stable infection with retroviral shRNA vectors. Knockdown was assessed through qRT-PCR for VEGFR1 and Western blot for VEGFR2. Cell morphology was assessed by optic microscopy. Cell numbers were assessed by cell counts at 48 hours in full growth media (FM) and under cell stress conditions (1% FM); proliferation was assessed by BrdU incorporation and apoptosis was assessed by a cell death Elisa in 1%FM. RESULTS: Knockdown of VEGFR1 was seen in 34% of CP-C, 22% of CP-D, and 0% of CP-B. Thus, we used CP-C only to assess effects of VEGFR1 knockdown. By Western blotting, we observed knockdown of VEGFR2 in CP-B and in CP-D, but not in CP-C. Thus, we used CP-B and CP-D to assess effects of VEGFR2 knockdown. We did not observed any morphology changes in the VEGFR knockdown cell lines compared to controls. In FM, dysplastic cells containing either VEGFR1 or VEGFR2 knockdown had higher cell numbers compared to control cells. In 1%FM, there was no significant difference in cell number between CP-C cells containing VEGFR1 knockdown and controls. In contrast, at 48 hours in 1%FM, CP-B and CP-D containing VEGFR2 knockdown had lower cell numbers (19.9 ± 1.3 X 104 and 10.5± 1.4 cells, respectively) compared to controls (41.4 ± 4.4 and 16.9 ± 1.6 cells). Compared to controls, VEGFR2 knockdown significantly increased BrdU incorporation in CP-B cells, even though overall cell number was decreased, whereas in CP-D, BrdU incorporation was decreased (p=0.059) along with overall cell number. Compared to controls, VEGFR2 knockdown increased apoptosis in CP-B and CP-D cells. CONCLUSIONS: VEGFR2, but not VEGFR1, contributes to cell growth of dysplastic Barrett's cells, but only under conditions of cell stress, with both pro-survival and pro-proliferative effects. These findings support a potential role for anti-VEGFR2 therapies in the treatment of high grade dysplasia in Barrett's esophagus.Item Intracellular Trafficking of Influenza Hemagglutinin and Members of the Low Density Lipoprotein Receptor Family(2004-12-15) Tall, Renee Danielle; Lehrman, Mark A.Polarized epithelial cells usually line body cavities, providing a barrier between two dissimilar environments. Distinct apical and basolateral membrane surfaces are maintained by sorting proteins in the biosynthetic and endocytic pathways. Influenza hemagglutinin is sorted to the apical membrane in polarized epithelial cells by a mechanism mediated by an association with lipid microdomains known as lipid rafts. Lipid rafts and their associated proteins are operationally defined by their resistance to detergent solubilization at cold temperatures. By systematic mutagenesis of the transmembrane domain of hemagglutinin, I show that ten consecutive amino acids are required to confer resistance to detergent extraction. Although some of the hemagglutinin transmembrane mutants were sorted apically without incorporation into detergent-resistant membranes, I determined that these mutants were transiently associated with lipid rafts. A small fraction of hemagglutinin coprecipitates with MAL/VIP17, a protein required for apical transport. The hemagglutinin and MAL that co-precipitated were contained in a detergent-resistant vesicle in an orientation consistent with a transport intermediate, suggesting that MAL might sort hemagglutinin into apical vesicles in the Golgi. However, the time course of the association of hemagglutinin and MAL in the biosynthetic pathway indicate that the two proteins do not associate until the majority of the HA reached the cell surface. Both the timing and limited extent of coprecipitation suggest that MAL may not sort hemagglutinin into apical vesicles in the biosynthetic pathway. Megalin, a member of the low density lipoprotein family of receptors, is sorted apically in polarized epithelial cells via a sorting signal present in its cytosolic domain. I show that the cytosolic domain of megalin associates with sorting nexin 17, a protein that interacts with all core members of the low density lipoprotein receptor family. Although sorting nexin 17 may not sort megalin apically in the biosynthetic pathway, I show that overexpression of sorting nexin 17 increases the rate of low density lipoprotein receptor recycling to the plasma membrane. The increase in recycling does not affect the expression of low density lipoprotein receptor at the cell surface, suggesting that sorting nexin 17 decreases the cycling time of the receptor.Item Isoflurane Preserves Viability of Highly Metabolic Renal Epithelial Cells Exposed to Anoxia(2016-04-01) Mantravadi, Vasudha; Kojima, Koji; Ambardekar, Aditee; Lin, Xihui; Gingrich, KevinBACKGROUND: Cells subjected to ischemia, whether in the context of hypoxia, hypovolemia, or circulatory collapse, undergo damage and death as a result of oxygen deprivation. Previous studies have shown that general anesthetics can protect cells from ischemic injury by lowering their aerobic metabolism and decreasing production of toxic metabolites, among other mechanisms (1, 2, 3). This very preliminary study investigated the potential protective effect of isoflurane on the survival of cells that have a fairly high baseline metabolic rate, human renal proximal tubular epithelial cells (HK-2) and human microvascular endothelial cells (HMEC), in an anoxic environment. METHODS: Cultured HK-2 and HMEC cells were incubated in a Forma Scientific Anaerobic System at 37C either in the absence (control) or presence (experimental) of 5% isoflurane for 0, 24, 48, 72, and 96 hours. Cell viability and metabolic activity were then assessed using live/dead fluorescence imaging and an MTT cell metabolism assay, respectively. RESULTS: In vitro exposure of cells to anoxia without isoflurane over a period of 96 hours, resulted in a reduction of viability of HK-2 cells from a baseline of 98%, to approximately 8-9%. Over the same period of time, viability of cells exposed to isoflurane and anoxia decreased to 35%. This represented a fourfold increase in survival of HK-2 cells exposed to isoflurane at 96 hours. At earlier time points, both cell death in anoxia, and the protective effect of isoflurane were less dramatic. HMECs did not undergo significant cell death upon exposure to either anoxia or anoxia with isoflurane, with 98% of the cells surviving the exposure to anoxia in both cases. The net metabolic activity, as assessed by absorbance using the MTT assay, decreased in HK-2 cells over increasing periods of anoxia, a trend that did not change with the addition of isoflurane. Metabolic activity of HMECs remained intact and relatively stable throughout the course of anoxic exposure. CONCLUSION: In this preliminary study, continuous exposure of HK-2 cells to 5% isoflurane during anoxic incubation had a protective effect on cell viability over a period of 96 hours. Whether this effect was also present in the less metabolically active HMECs, was not determined, as anoxia over the time period of the study had little effect on cell viability in either the experimental group or in the control group. The protective effect observed for HK-2 cells will likely vary with differences in metabolic requirements of different cell types, types and concentrations of anesthetic agents, and duration of anesthetic exposure. Anesthetic treatment may need to be tailored specifically to a cell type to confer the protective effects desired.Item The Ligand and Function of the RegIII Family of Bactericidal C-Type Lectins(2006-08-11) Cash, Heather Lynn; Hooper, Lora V.Beginning at birth, the intestines of humans and other mammals are colonized with a diverse society of resident bacteria that play a crucial role in host nutrient metabolism. To maintain this commensal relationship, resident microbes must be prevented from crossing the intestinal epithelium into host tissues where they can cause inflammation and sepsis. The innate immune system plays a crucial role in preventing bacterial incursions across gut epithelial surfaces. Mucosal epithelial cells produce a variety of secreted antimicrobial proteins that help to prevent bacterial attachment and encroachment at epithelial surfaces. Among these, Paneth cells are specialized small intestinal epithelial cells that have been shown to produce and secrete antimicrobial proteins and peptides. To gain new insights into the adaptation of mucosal surfaces to microbial challenges, the Hooper lab has used DNA microarrays to screen for Paneth cell genes whose expression is modulated by intestinal microbes. This screen revealed that expression of two C-type lectins, RegIIIbeta and RegIIIgamma , is strongly induced following intestinal colonization with resident microbes. Two features suggested that members of the RegIII family may have microbicidal functions. First, they are C-type lectin family members. Other C-type lectins, including the mannose binding lectin, have well-characterized innate immune functions and play critical roles in microbial killing by recruiting complement. Second, I have shown that the murine RegIII lectins localize to intestinal crypt cells, including Paneth cell secretory granules, and that they bind to luminal bacteria harvested from intestinal conditions. Based on these observations, we hypothesized that this family of proteins may perform an innate immune function, specifically antimicrobial defense. The studies reported in this thesis characterize a family of C-type lectins. Specifically, we determined that these proteins interact with peptidoglycan by binding with high affinity to its glycan structure, representing a unique blend of peptidoglycan recognition and lectin function. Additionally, we have demonstrated that this binding results in the specific disruption of the Gram positive bacterial cell wall, where peptidoglycan is exposed, which is the first example of a family of directly bactericidal C-type lectins. We also present evidence for the regulation of these bactericidal proteins by colonization with an intestinal microflora. Therefore, the research presented in this thesis elucidates the function of three members of the RegIII family, in both mice and humans.Item Mechanical Regulation of Glycolysis via Cytoskeleton Architecture(2019-11-14) Park, Jin Suk; Bachoo, Robert; Danuser, Gaudenz; DeBerardinis, Ralph J.; Shay, Jerry W.The mechanical properties of the microenvironment continuously induce cells to modulate functions like growth, survival, apoptosis, differentiation, and morphogenesis. These adaptations rely on dynamic cytoskeletal remodeling and actomyosin contractility. Although all these processes are coupled to energy consumption, it is unknown if and how cells metabolically adapt to mechanical cues. In this thesis, I demonstrate that phosphofructokinase (PFK), a rate-limiting regulator of glycolysis, responds to mechanical cues in human bronchial epithelial cells (HBECs). Transferring HBECs from stiff to soft substrates causes downregulation of glycolysis via degradation of PFK. The loss of PFK expression is triggered by stress fiber disassembly, which releases the PFK-targeting E3 ubiquitin ligase, tripartite motif(TRIM)-containing protein 21 (TRIM21). Transformed non-small cell lung cancer cells (NSCLCs), which maintain high glycolytic rates regardless of changing mechanical cues, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 to a stress fiber population that is insensitive to substrate stiffness. Thus, I dissected a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to modulate energy production in variable microenvironments, while the resistance of the cytoskeleton to respond to extracellular mechanical cues allows high glycolytic rates to persist in cancer cells despite constant alterations of the tumor tissue.Item Mitochondrial Dynamics in Renal Cell with Burn Serum Stimulation: An Observational Study(2017-01-17) Maxwell, Christian T. W.; Huebinger, Ryan M.; Song, Juquan; Sehat, Alvand J.; Wolf, Steven E.BACKGROUND: Myoblasts and myocardiocytes have increased cell death with burn serum stimulation, which is associated with mitochondrial fission and function impairment. Acute kidney injury is a significant issue in burn patients; however, the role of the mitochondrial response in renal cells (RCs) has not been explored. The purpose of this summer project was to establish a method that observes mitochondrial dynamics in renal epithelial cells upon exposure to burn serum. HYPOTHESIS: The mitochondrial fission/fusion cycle in human RCs is disrupted following exposure to burn serum. METHODS: Human primary renal proximal tubule epithelial cells were cultured with Renal Epithelial Cell Growth Media in an incubator at 37°C with 5% CO2. Upon reaching 65-70% confluency, RCs were treated with growth media containing 10% rat serum (RS) either from control rats or 40% total body surface area scald burn rats. 3nM of MitoTracker Green FM dye was added to all cell treatments to stain the mitochondria (MT). Live cell images were taken under a Nikon Ti Eclipse Confocal microscope at 6, 24, 48, 72, and 96 hour time points; subsequently, images were analyzed with Nikon software to identify fluorescent intensity, mitochondrial volume and elongation. T-tests were then applied to analyze the significance with Bonferroni correction. Significance after correction was determined if p < .01 . OUTCOMES: RC morphology was cuboidal and refractile in the culture media. During serum stimulation experiments, the MT of RCs gradually increased in fluorescent intensity and maintained a rod-like shape in both groups. Specifically, the MT exposed to normal RS had significantly higher intensity at the 72 and 96 hour marks than those in burn RS. Despite these results, cell viability was determined to be 48 hours, since 60% or more of cells had detached at the 72 and 96 hour marks, indicating cell death. There were no significant differences in elongation and volume between burn and normal serum stimulation at each time point. CONCLUSIONS: The cell culture protocol for human primary renal epithelial cells was established this summer. Although rats and humans share evolutionarily conserved traits, the RS did not significantly influence the more evolved human RC.Item Protecting Healthy Cells Against the Negative Effects of Radiation Therapy for Lung Cancer(2017-03-06) El-Ashmawy, Mariam A.; Burma, Sandeep; DeBerardinis, Ralph J.; Mendelson, Carole R.; Shay, Jerry W.; Wright, Woodring E.Although radiation therapy is a commonly used treatment for many human diseases including cancer, ionizing radiation produces reactive oxygen species that can damage both cancer and healthy cells. Synthetic triterpenoids, including CDDO-Me, act as anti-inflammatory, antioxidant modulators primarily by inducing the Nrf2/ARE pathway. In the documented series of experiments, I show that CDDO-Me can be used as a radioprotector in normal non-cancerous human lung and breast epithelial cells, whereas CDDO-Me does not further protect cancer lines from radiation-induced cytotoxicity, nor does it protect experimentally transformed human bronchial epithelial cells with progressive oncogenic manipulations. Additionally, CDDO-Me protects human lymphocytes against radiation-induced DNA damage. As part of these studies other compounds (RTA-408, TA-65, and Yel-002) were also tested for radioprotective effects in epithelial and cancer cells as well as human lymphocytes. A therapeutic window exists in which CDDO-Me protects normal cells from radiation by activating the Nrf2 pathway, but does not protect experimentally transformed or cancer cell lines. This suggests that use of this oral available, non-toxic class of drug can protect non-cancerous healthy cells during radiotherapy, potentially resulting in better outcomes with less toxicity for patients. The effects of radiation and DNA damage in traditional two-dimensional (2D) cell culture conditions in vitro may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture systems since important signals, provided by the extracellular matrix and microenvironment, are lost in 2D monolayer cultures. While irradiating premalignant HBECs in traditional 2D culture significantly increased cancer progression phenotypes, irradiation in 3D culture reduced radiation-induced transformation compared to 2D. Furthermore, 3D cell culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation--implying there was no obvious selection against any cells in or dissociated from 3D conditions. My findings indicate that culture conditions are crucial for cellular responses to radiation and can affect cancer progression. If 3D culture is a more biologically representative model compared to 2D cultures, then current studies assessing transformation and radiation may be overestimating radiation risks using standard 2D culture methods.Item With No Lysine 1 (WNK1): A Potential Regulator of The Lysosomal Degradation Pathway(2007-05-22) Lenertz, Lisa Yvonne; Cobb, Melanie H.With no Lysine (K) 1 (WNK1) is an atypical serine/threonine protein kinase that has its catalytic lysine positioned in a unique location. This kinase, along with another member of the WNK family, WNK4, has been genetically linked to pseudohypoaldosteronism type II (PHAII), which is characterized by both hypertension and hyperkalemia. Several groups have used reconstitution assays in Xenopus oocytes and mammalian cell lines to show WNKs regulate the surface expression and/or activity of various ion transporters and channels, including the epithelial sodium channel (ENaC) and the sodium chloride co-transporter (NCCT). Although the mechanisms for regulating these cell surface proteins are not well defined, it appears that WNKs may modulate the intracellular trafficking of these channels and transporters. To help define the mechanisms WNK1 utilizes to influence blood pressure and to characterize this kinase biochemically, I performed a WNK1 kinase activation screen and a WNK1 yeast-two-hybrid screen. I have shown that WNK1 kinase activity increases in response to osmotic stress, which may imply its kinase activity is important for regulating ion homeostasis in response to a change in cell volume. I have also shown that a proline-rich region of WNK1 interacts with vacuolar protein sorting 4a (VPS4a), an ATPase that helps sort cargo from the plasma membrane to lysosomes. Cells expressing a VPS4 ATP-hydrolysis mutant trap cargo from the cell surface in an aberrant endosomal structure, slowing protein degradation via the lysosomal pathway. I hypothesize that WNK1 delivers cargo to VPS4a to facilitate the degradation of plasma membrane proteins.