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Intestinal inflammatory disorders such as Crohn’s disease contribute significantly to human mortality and morbidity. Although the cells and molecules involved in suppression of intestinal inflammation have been extensively documented in mouse models, a full understanding of how these work together in the healthy and diseased gut remains elusive. It is known, however, that tight regulation over TH17 cells and regulatory T cells (Treg) is required to maintain immune homeostasis in the intestine. Activation of the cytokine transforming growth factor-β (TGFβ), which is secreted by immune cells as an inactive complex, plays a crucial role in the induction of both Treg and TH17 cells. Recent work has shown that the αvβ8 integrin is required for activation of TGFβ by murine dendritic cells (DC). Murine integrin αvβ8 is thus of fundamental importance for Treg and TH17 induction and, subsequently, for intestinal immune homeostasis. However, little is known about the signals controlling the expression of integrin αvβ8 on intestinal DC. Furthermore, whether this system is also important for regulation of the human system is entirely unknown. Here, expression of integrin αvβ8 is shown on human intestinal CD1c+CD103+SIRPα+ DC and CD1c+CD103-SIRPα+ DC, but not on CD141+CD103+SIRPα- DC. Expression of integrin αvβ8 is increased on DC from Crohn’s disease patients, and on DC from non-IBD donors treated with LPS. Both LPS and a number of probiotic bacteria were also able to induce integrin αvβ8 expression on human monocyte-derived DC (moDC), which suggested that the microbiota may play a role in the regulation of human integrin αvβ8. Importantly, we have also shown that TGFβ is activated by integrin αvβ8 on human moDC, and that integrin αvβ8 promotes expression of forkhead box P3 (FOXP3) in naïve human T cells in vitro. Together, these data suggest that integrin αvβ8-mediated activation of TGFβ by DC may play an important role in the regulation of human T cell responses in the human intestine, and that this pathway may be perturbed during intestinal inflammation.

As retinal pigmented epithelium (RPE) cells are passaged, they undergo an irreversible epithelial-to-mesenchymal transition (EMT). We have shown previously that growth differentiation factor 6 (GDF6), a member of the transforming growth factor-beta (TGFβ) family, is highly upregulated in RPE cells that have lost the capacity to obtain an epithelial phenotype. We hypothesize that GDF6 plays an integral role in the irreversible transition of an RPE cell from an epithelial state to a mesenchymal state.To test this hypothesis, we overexpressed GDF6 in differentiation competent RPE and assessed the effects on phenotype and gene expression. To evaluate what receptors and signaling pathways might mediate GDF6’s effects, cells were also treated with an Alk5 inhibitor (Repsox), an Alk2/3/6 inhibitor (LDN-193189), or both in combination. Passage 0 RPE transduced with GDF6 produce significantly less pigmentation than cells infected with an empty vector control. This reduction in pigmentation is accompanied with a change in cell morphology; the control cells maintain a cuboidal morphology and the GDF6 expressing cells have a spindle-like morphology. Quantitative PCR analysis reveals that RPE cells transduced with GDF6 significantly upregulate known EMT markers like ACTA2, CTGF, and COL1A1 and downregulate classical RPE markers such as LRAT and PMEL compared to control cells. Both RepSox and LDN-193189 have the ability to reverse the GDF6 phenotype. While the phenotype is recovered, RNA-seq analysis reveals GDF6-mediated regulation of genes that are unaffected by inhibitors, such as TGFβ1, MSX2, and CDH1. GDF6 is involved in the EMT process in RPE. We believe GDF6 upregulates TGFβ1, which in turn promotes EMT. The ability to revert back to an epithelial cell is inhibited by GDF6. Cells exposed to GDF6 will prematurely undergo EMT, simultaneously downregulating traditional RPE markers while upregulating EMT and wound response markers. Inhibition of the BMP receptors rescues the GDF6 phenotype, therefore inhibition of GDF6 may be integral in prolonging the integrity and functional lifespan of the RPE. As such, inhibition of GDF6 may help restore RPE to their epithelial state in diseases like proliferative vitreoretinopathy and age-related macular degeneration.

Epithelial-mesenchymal transition (EMT) is a conserved cellular plasticity program that is reactivated in carcinoma cells and drives metastasis. EMT is well studied, but its regulatory mechanisms remain unclear. In this dissertation, we explore two ways in which the ubiquitin-proteasome system regulates EMT. First, we investigate the role of the 26S proteasome in regulating EMT. In Chapters 1-2, we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy.Second, we explore potential novel regulators of EMT within the ubiquitin-proteasome system pathway. In Chapters 3-4, we uncover novel regulators of EMT by mining previously published microarray data and found a group of deubiquitinases (DUBs) upregulated in cells that have undergone EMT. Here, we show that one DUB in particular, Ubiquitin specific peptidase 11 (USP11), enhances TGF-β-induced EMT and self-renewal in immortalized human mammary epithelial cells. Furthermore, modulating USP11 expression in human breast cancer cell lines altered migratory capacity in vitro and metastasis in vivo. Moreover, we found that high USP11 expression in human breast cancer patient clinical samples correlated with decreased survival. Mechanistically, modulating USP11 expression altered the stability of TGF-β receptor type 2 (TGFBR2) and TGF-β downstream signaling in human breast cancer cell lines. Together, these data suggest that deubiquitination of TGFBR2 by USP11, effectively spares TGFBR2 from proteasomal degradation to promote EMT and metastasis and suggest USP11 as a potential therapeutic target for breast cancer.

INTRODUCTION: Stem cells have become promising treatments for osteoarthritis due to the cells’ ability to regenerate cartilage and availability from bone marrow. Various studies have established the chondrogenic potential of human marrow stromal cells (hMSCs) upon treatment with transforming growth factor β1 (TGF-β1), yet the difference in potential between cells derived from young subjects and those derived from elder subjects has not been confirmed. OBJECTIVES: This study seeks to establish whether the chondrogenic potential of hMSCs changes with age and sex. This study used a high-density 2D model to measure the acute response of hMSCs to chondrogenic induction over a short time course and various treatment levels. The experiments investigated the expression of chondrogenic genes and expression of TGF-β1 receptors (ALK5) in hMSCs after TGF-β1 treatment to determine whether pediatric hMSCs have more potential for chondrocyte differentiation than adult hMSCs. METHODS: With IRB approval, nine bone marrow samples were obtained from discarded tissue of adults undergoing total hip replacement and juveniles requiring bone graft for alveolar cleft repair. Subject ages ranged from age 8 to 66. Low-density mononucleated cells were cultured in plastic tissue culture dishes. Adherent hMSCs were expanded in monolayer culture with phenol red-free α-MEM medium with 10% fetal bovine serum. After 48 hours of treatment with TGF-β1, cells were collected for RNA extraction and RT-PCR analysis of chondrogenic genes and TGF-β1 receptor levels. Alcian blue staining in 24-well plates of hMSCs was performed after 10 days to compare the effects of different concentrations of TGF-β1, and the effects of another inducer of chondrogenesis, kartogenin (KGN) on matrix accumulation. RESULTS: Gel electrophoresis of PCR products revealed no consistent trend in chondrogenic mRNA expression in pediatric cells compared to adult cells, or female cells compared to male cells. The data indicate that the change in chondrogenic potential of hMSCs with age and sex is inconsistent. KGN showed no consistent effect on hMSCs. Cells with high baseline levels of TGF-β1 receptor (ALK5) showed no upregulation of ALK5 after TGF-β1 treatment, while samples with low basal expression of TGF-β1 receptors showed upregulation after TGF-β1 treatment. CONCLUSIONS: There is still much debate in the literature regarding the potential of adult hMSC chondrogenesis compared to juveniles. This study confirms the irreproducibility of displaying differences between young and adult hMSCs. A larger sample size is needed to establish a correlation between age and chondrogenic potential. Further in vitro studies will consider the optimum time course and concentration of TGF-β1 to observe differences in gene expression of cells, and will identify other clinical determinants of differentiation potential.

Ribosomes are large complex molecular machineries required for the synthesis of proteins via a process called protein translation. The importance of this molecular machine is conserved and is found within all living cells. Two subunits (designated the small and large subunits) form a functional ribosome. Each subunit is a complex of ribosomal RNA and a variety of proteins. Within the ribosome, amino acids are linked together depending on the order specified by the mRNA molecule. Due to the importance of ribosomes in maintaining cellular function, one would expect defects in ribosome function, or biogenesis, to have a systemic effect on the organism. However, what is typically seen with disorders involving aberrant ribosome functions, a.k.a. ribosomopathy, is a tissue-specific defect. Specifically, patients normally present themselves with bone marrow failure, which ultimately leads to severe anemia. Other common features in patients include a shorter stature and a higher predisposition to certain cancers. Diamond Blackfan anemia (DBA) is the first described ribosome-associated disorder with ribosomal protein S19, RPS19, being the most commonly mutated gene found in patients. Since then, the number of ribosomal proteins found mutated in DBA patients has expanded and includes genes such as RPL11, RPL5, and RPS24. Another ribosomopathy is 5q-syndrome. In this disease, a large chromosomal deletion is found in patients. Within the commonly deleted region (CDR), it is believe that ribosomal protein S14, RPS14, is the candidate gene associated with the macrocytic anemia seen in the disease.The effects of p53 signaling during ribosome stress have been well documented in both patient bone marrow and in models of ribosomopathies. Aberrant ribosome function has been shown to increase p53 signaling through stabilization of p53 through a RP-MDM2-p53 checkpoint mechanism in the cell. Upon increases in p53 activity, p53-mediated cell cycle arrest and apoptosis occurs. Reversal of this increase in p53 signaling has been shown to relive the phenotypic and anemic defects in cellular and animal models. However, the use of p53 inhibition towards patient care has been cautioned due to risk of cancer progression. p53 is a major tumor suppressor gene in the cell and abolishing its activity has been shown to elevate cancer risk. Patients with malfunctioning ribosome proteins are at a higher risk of tumor development; therefore, targeting p53 may further enhance tumor risk. This dilemma raises the need for novel therapeutics in tackling the treatment of ribosomopathies and to further understand disease pathology.Animal models are indispensible for understanding the molecular mechanism and pathology of a disease. Zebrafish is an attractive vertebrate model to use in biological studies due to its rapid development, small size, transparent nature, and ease of manipulating gene function. Furthermore, there are many conserved genes between mammals and zebrafish, thereby making the model useful for understanding gene function. The advent of genome-editing technologies applied to the zebrafish model (i.e. TALEN, CRISPR/Cas9) has expanded the use of the zebrafish to model human diseases. In this work, we model two ribosome-associated disorders in the zebrafish, DBA and 5q-sydrome, via targeted RPL11 and RPS14, respectively. Using these models, we show that a delay in late-stage erythropoiesis occurs and is followed by cell death upon ribosome stress. The delay in late-stage erythropoiesis is independent of p53 signaling and may, in part, be due to overexpression of Lft1. We demonstrated the ability of RAP-011, an activin receptor type IIA ligand trap, to increase erythropoiesis in the zebrafish and restore erythroid levels in our zebrafish models. Furthermore, the effects of RAP-011 are independent of p53 signaling, and thus, offer a new strategy in the treatment of ribosomopathies.

Renal blood flow and micropuncture studies were conducted in late gestation fetal sheep (gestational age 134 - 141 days; term 150 days) and neonatal lambs (8 - 18 days after birth) to study the forces involved in glomerular filtration (GFR) and characterize the tubuloglomerular feedback (TGF) system during development. These studies required the kidney to be immobilized so stable models in acutely prepared anaesthetized animals were developed. Fetuses were studied in a heated water bath exteriorized from the uterus but with an intact umbilical circulation. The lower GFR in fetuses than lambs was found to be due to both lower net filtration pressures (P

Airway remodeling observed in chronic inflammatory diseases of the respiratory system is characterized by mucous cell hyperplasia, smooth muscle cell proliferation and subepithelial fibrosis. Interleukin (IL)-13 is a central mediator in the pathogenesis of inflammatory airway diseases playing a significant role in epithelial remodeling. In this study, we examined mechanisms through which IL-13 induces proliferation of normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture in vitro, a model that allows for the maintenance of differentiated structure and function. It was found that IL-13-induced proliferation of NHBE cells is mediated by the autocrine/paracrine action of transforming growth factor-a (TGFa) that is produced by the epithelial cells and subsequently binds to the epidermal growth factor receptor (EGFR) on these cells. IL-13-induced release of TGFa involves the rapid mobilization of intracellular stores of TGFa to the apical cell surface, where membrane bound TGFa is cleaved by tumor necrosis factor-a converting enzyme (TACE), resulting in released growth factor. Both neutralizing anti-TGFa antibodies and the EGFR tyrosine kinase inhibitor AG1478 block IL-13-induced proliferation in concentration-dependent manners. Additionally, IL-13 induces the activation of the mitogen activated (MAP) kinase pathway and the phosphatidylinositol 3-kinase (PI3K) pathway, both of which are required for IL-13-induced proliferation. However, activation of the MAP kinase pathway by IL-13 does not appear to be TGFa mediated, as neutralizing anti-TGFa antibodies and EGFR tyrosine kinase inhibitors have little effect on IL-13-induced MAP kinase. PI3K was found to be involved in IL-13-induced release of TGFa as the specific inhibitor of PI3K, LY294002, blocks release of the growth factor. In summary, IL-13-induced proliferation of NHBE cells involves the intracellular redistribution and subsequent TACE-mediated release of TGFa, an event mediated by PI3K. This proliferation then involves the interaction of TGFa with the EGFR and requires activation of both the MAP kinase and the PI3K signaling cascades. These data establish a mechanistic framework for further study into the effects of IL-13 on bronchial epithelial cells as well as additional cell types that may interact with IL-13, or growth factors induced by this cytokine, in an in vivo setting.