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Articular cartilage lesions are often caused by joint trauma and can progress to osteoarthritis (OA) if left untreated. Cartilage tissue engineering is a promising approach for chondral lesion repair, involving the cultivation of cell-seeded scaffolds to generate neocartilage tissues recapitulating composition, structure, and function of native cartilage. Transforming growth factor beta (TGF-β) is widely utilized in cartilage tissue engineering for its ability to promote chondrogenesis and extracellular matrix (ECM) biosynthesis. Conventionally, TGF-β is supplemented in culture medium at supraphysiologic doses (10-100 ng/mL) during in vitro cultivation to regenerate neocartilage with native-matched sGAG content and mechanical properties. However, these doses are 10-1000-fold higher than the physiologic range, promoting undesirable tissue features that are detrimental to the functional behavior of hyaline cartilage. Additionally, TGF-β gradients from media supplementation can induce pronounced heterogeneities in ECM distribution, potentially compromising the survival of engineered cartilage under physiologic loading. The dissertation aims to enhance cartilage regeneration quality using bio-inspired latent TGF-β (LTGF-β) conjugated scaffolds. We hypothesize that LTGF-β scaffolds can achieve uniform delivery of moderated, near-physiologic doses of TGF-β through cell-mediated activation, inducing homogeneous and more hyaline cartilage-like tissue growth. We first evaluated the impact of physiologic TGF-β doses on tissue growth. To address issues related to TGF-β concentration gradients and tissue heterogeneities, we employed a reduced-size construct model. Our findings demonstrate that physiologic doses of TGF-β promote significant enhancements in tissue properties for reduced-size tissues, while also mitigating undesirable outcomes associated with excessive TGF-β. Subsequently, we developed bio-inspired LTGF-β-conjugated scaffolds to deliver physiologic doses of TGF-β. We established a quantification platform based on TGF-β autoinduction to accurately measure the bioactivity level of delivered TGF-β, confirming conjugated LTGF-β can be activated in physiologic range. Further, this quantification platform exhibits versatility for applications in native tissue studies and other TE platforms. Lastly, we determined that LTGF-β conjugation led to enhancements in tissue functional properties comparable to native tissue, while mitigating the abnormal features of neocartilage associated with TGF-β excesses. Moreover, LTGF-β conjugation significantly improves tissue spatial homogeneities in composition and mechanical properties, offering promising implications for enhancing clinical regeneration outcomes.

Advancements in therapy have improved remission and prolonged survival for patients with B cell malignancies. Unfortunately, for many challenging cancer subtypes such as mantle cell lymphoma, most patients will still relapse and die from the cancer. Chimeric antigen receptor (CAR)-T cell therapy uses genetically modified T cells expressing CAR protein to recognize and kill cancers expressing a specific antigen, such as CD19. Although CD19 CAR-T therapy has been very effective against relapsed/refractory B cell cancers, antigen escape and relapse still occur in up to 40% of patients treated with CD19 CAR-T. This work describes the creation and validation of a novel, BAFF ligand-based CAR that aims to prevent antigen escape by being able to bind multiple BAFF receptors expressed by the cancer cell, rather than one antigen alone. Using cytotoxicity assays, we demonstrate the functionality and specificity of BAFF CAR-T cells in killing several types of B cell malignancies, and we further confirm their efficacy in several mouse models of cancer. These results have helped bring BAFF CAR-T therapy to Phase I clinical trials, and we hope they prove efficacious for patients with relapsed/refractory B cell malignancies.Meanwhile, interest has rapidly increased in natural killer (NK) cell-based immunotherapies. NK cells have important roles in cancer immunosurveillance and offer advantages over T cells in clinical safety and time-to-treatment. However, NK therapies face challenges in efficacy and persistence, often due to the presence of TGF-β, a powerful immunosuppressive cytokine that is frequently elevated in cancer patients and a primary cause of NK cell dysfunction. In this work, we characterize an undiscovered role of cyclin dependent kinase 5 (Cdk5) and its coactivator p35 in NK cell cytotoxicity. Using genetic tools to modulate Cdk5/p35 kinase activity in human NK cells, as well as using NK cells from p35 knockout mice, we show that Cdk5/p35 negatively regulates NK cytotoxicity. Furthermore, we show that TGF-β induces p35 expression in NK cells, while p35 knockdown or expression of dominant negative Cdk5 resists TGF-β-mediated inhibition of NK cytotoxicity. Further understanding of these mechanisms may reveal novel strategies to enhance NK-based immunotherapies and overcome their dysfunction in cancer patients.

Orthodontics is a specialty of dentistry that focuses on the correction of malpositioned teeth and jaws through dentofacial orthopedics. Thus, a critical understanding of tooth movement, retention, and relapse is crucial for clinical practice and treatment advancements for our patients. The foundation of orthodontic tooth movement (OTM) and relapse must be studied at the cellular level, by examining the role of osteocytes. Many studies have explored the mechanisms underlying OTM and relapse but have focused more on the roles of osteoclasts and osteoblasts. Recent studies are now investigating osteocytes and unveiled that these cells are integral in mechanical-sensing in bone. On the molecular level, one of the pathways that is warranting further investigation is TGF-ß signaling in osteocytes with respect to orthodontic tooth movement. Studies showing that osteocytes directly remodel their perilacunar/canalicular matrix suggest that TGF-ß controls bone quality through this remodeling as well. TGF-ß has also been shown to stimulate osteocytes to increase RANKL production, ultimately leading to osteoclastogenesis in long bones. The aim of this study is to determine the extent to which osteocyte-intrinsic TGF-ß signaling in alveolar bone affects orthodontic tooth movement rate and alveolar bone remodeling. We will employ TβRIIocy-/- mice to examine the mechanism of TGF-β signaling in osteocytes. TβRIIocy-/- and control littermates will undergo OTM for 14 days through a spring assembly mesializing the upper left first molar. MicroCT scanning will be used to measure factors such as orthodontic tooth movement rate, bone volume, bone mineral density, alveolar bone height, root length, and root volume. The goal of this project is to analyze osteocyte function during orthodontic tooth movement and understand its mechanism so that we can safely and efficiently move teeth while reducing dental relapse potential afterwards. Thus, we can spark future clinical applications in orthodontic treatment and efficiency.

Cardiomyocytes and macrophages have been found to interact via connexin-43 hemichannels. The role of connexin-43, however, is not fully understood. This study shows that these interactions aid in increasing the membrane potential of cardiomyocytes allowing contraction of the cells. HL-1 cardiomyocytes and RAW 264.7 macrophages in coculture increased expression of connexin-43 compared to cardiomyocytes alone. Co-cultures also increased the fluorescence of Di-8-ANEPPS potentiometric dye indicating an increase in cardiomyocyte membrane potential. Treatment with IL-10 and TGF-beta further increased connexin-43 expression and membrane potential. Treatment with SOCS3 inhibited the effects of TGF-beta and IL-10 while having no effect on its own. Confocal imaging can provide visual evidence of these interactions with image layering, 3D imaging and enhanced color.

Polycystic Ovary Syndrome (PCOS) is primarily thought of as a reproductive condition, although it has profound effects on metabolic health, with 38-80% of women with PCOS presenting with insulin resistance. This underlying insulin resistance occurs independently of obesity and has negative effects on other features of the condition. The identification of peripheral insulin resistance in PCOS occurred in 1989, and despite several advances in this area, the molecular mechanisms responsible for peripheral insulin resistance in women with PCOS remain unclear. In addition, interventional studies from women with PCOS have identified impaired responses to insulin-sensitizing therapies. A possible explanation for the underlying insulin resistance and poor response to insulin-sensitizing treatments may be the dysregulation of Transforming Growth Factor-beta (TGF-beta) signalling. The dysregulation of TGF-beta signalling is responsible for remodelling in the ovarian tissues and reproductive dysfunction. Due to the systemic action of TGF-beta signalling, it may be conceivable that these effects extend beyond the reproductive tissues to the peripheral tissues. This presents a possible mechanism that could contribute to the development of insulin resistance in women with PCOS. Understanding the underlying mechanisms of insulin resistance will allow for improvements in diagnosis and for targeted therapies to be developed, which are currently lacking for women with PCOS. The overall aim of this thesis was to determine if dysregulated TGF-beta signalling plays a role in skeletal muscle insulin resistance and can influence metabolic responses to exercise in women with PCOS. This was achieved through a combination of in vivo and in vitro studies focusing on skeletal muscle metabolism. Study 1 explored the role of TGF-beta ligands, TGF-beta 1 and Anti-müllerian hormone (AMH), on glucose uptake and insulin signalling in myotubes from women with PCOS and healthy controls. In line with previous studies, we showed that in vivo metabolic phenotype was not accurately retained in cultured myotubes from women with PCOS, suggesting that a combination of the in vivo environmental factors and intrinsic defects lead to the development of insulin resistance. TGF-beta 1 and AMH had distinct metabolic effects. TGF-beta 1 increased basal and insulin-stimulated glucose uptake, while AMH decreased glucose uptake and PI3K-p110 expression, which was accompanied by an increase in inhibitory IRS-1ser312 phosphorylation. Study 2 assessed the response of myotubes from women with PCOS and healthy controls to an in vitro model of contraction with and without TGF-beta 1 or AMH. It was found that myotubes from women with PCOS and healthy controls display minimal differences in exercise-induced signalling transduction. Myotubes from healthy women showed an increase in the phosphorylation of p38 MAPK and CREB, which appeared to be absent in the myotubes from women with PCOS. The TGF-beta ligands AMH and TGF-beta 1 do not appear to influence in vitro skeletal muscle exercise-like signalling responses. In Study 3, a cross-sectional approach was used to assess skeletal muscle TGF-beta and insulin signalling in women with PCOS compared to overweight and lean controls. Insulin sensitivity, as determined by euglycemic–hyperinsulinemic clamp, confirmed previous findings that women with PCOS have a significant reduction in insulin sensitivity compared to controls. This insulin resistance occurred in the absence of any identifiable defects in skeletal muscle insulin signalling and did not appear to be related to TGF-beta signalling. Women with PCOS had greater levels of basal phosphorylation of p38 MAPK, suggesting that excessive reactive oxygen species and/or inflammation may contribute to insulin resistance. In Study 4, overweight women with PCOS participated in a 12-week exercise training intervention. We aimed to determine if changes in insulin sensitivity following 12 weeks of moderate or high-intensity exercise training were related to aberrant TGF-beta signalling and collagen deposition in the skeletal muscle. Exercise training of high and moderate intensities resulted in improvements in insulin sensitivity and cardiorespiratory fitness, with the metabolic benefits being more noticeable following the high-intensity intervention. Improvements in insulin sensitivity occurred independently of changes in body composition, TGF-beta signalling and other clinical measures. Collectively, the results from these studies demonstrate the role of TGF-beta ligands and signalling dysregulation in skeletal muscle metabolism in vitro. However, these findings do not appear to translate in vivo where insulin sensitivity was not related to TGF-beta signalling in women with or without PCOS. There was no apparent evidence of dysregulation of TGF- beta signalling or insulin signalling in women with PCOS. Furthermore, improvements in insulin sensitivity following 12 weeks of high- or moderate-intensity exercise training occurred independently of changes in TGF-beta signalling.

Breast cancer is the most commonly diagnosed cancer in women, claiming the lives of more than 40,000 women each year. The majority of these cancer associated deaths are due to metastasis, a clinically incurable disease with few treatment options. The multifactorial cytokine, TGF-Β, functions to mediate epithelial mesenchymal transition (EMT), a critical process by which breast cancer cells metastasize. Following dissemination from the primary tumor, breast cancer stem cells (BCSCs), cells capable of tumor initiation, can lie dormant and non-proliferative for decades before emerging as distant metastases. The studies presented herein have identified a glycolytic gene, Pfkfb3, as a key mediator of TGF-Β induced EMT and as a driver of emergence from metastatic dormancy. We show that aberrant Pfkfb3 expression can mediate escape from metastatic dormancy, doing so through the upregulation of BCSC frequency. Furthermore, we show that inhibition of autophagy, a key cytoprotective mechanism employed by BCSCs, can promote aberrant Pfkfb3 expression and drive metastatic outgrowth following a period of metastatic dormancy. We have identified Pfkfb3 as an autophagy substrate, interacting with the UBA domain of p62. During periods of high autophagy, p62 bound Pfkfb3 is degraded. However, upon autophagy inhibition, p62 accumulation promotes the stabilization of Pfkfb3 which ultimately drives emergence from dormancy and overt metastasis. Dissemination of BCSCs to distant metastatic sites is mediated in large part through TGF-Β-stimulated EMT. We demonstrate that Pfkfb3 expression is critical for breast cancer invasion and metastasis both in vitro and in vivo. In addition to roles in mediating survival through EMT, we also show that Pfkfb3 can modulate autophagy activation, a key regulatory mechanism of TGF-Β stimulated EMT. Importantly, we demonstrate that autophagy inhibition promotes the stabilization of the critical EMT mediator, Twist, suggesting that autophagy can selectively degrade Twist during periods of autophagy activation. Taken together, these data suggest a critical role for Pfkfb3 in mediating metastasis and emergence from dormancy. Furthermore, the development of a therapeutic aimed at targeting Pfkfb3, perhaps in combination with other targeted therapies and/or chemotherapeutics, could provide significant clinical benefit for breast cancer patients.

Growth differentiation factor 8 (GDF8), a.k.a. myostatin, is a member of the activin subclass within the larger TGFß superfamily of signaling ligands that contains over 30 distinct members. Discovered in 1997, GDF8 quickly became characterized as a negative regulator of muscle mass because a highly active GDF8 caused muscle wasting or atrophy. In contrast, when GDF8 is rendered inactive through extracellular inhibitors or mutations, massive muscle gain was observed. This discovery generated massive interest within the pharmaceutical industry to manufacture inhibitors for GDF8 to combat a variety of muscle wasting diseases. A better understanding of how GDF8 is regulated in vivo is imperative to develop efficacious and novel inhibitors against GDF8. The activity of GDF8 is tightly regulated in vivo by several different processes. TGFß ligands are synthesized as large precursor proteins with a N-terminal signal sequence and prodomain followed by the mature signaling domain. The prodomain is cleaved from the mature domain, but unlike most other TGFß family members, GDF8 forms a high affinity interaction with its prodomain rendering it inactive or latent. Prior to our work, the molecular mechanisms dictating latency were not well understood but were hypothesized to be like the latent TGFß1 procomplex. Using the TGFß1 procomplex as a model, we identified residues critical for a stable latent GDF8 procomplex. Furthermore, the structure of the latent GDF8 procomplex was solved by another laboratory and our mutants could be further characterized to determine the molecular mechanisms of GDF8 latency. In order to signal, latent GDF8 needs to be activated by a member of the tolloid family of metalloproteases which proteolytically cleaves the prodomain. While the site of tolloid cleavage on the GDF8 prodomain has been identified, how tolloid recognized the prodomain as a substrate was unknown. Unlike other proteases, tolloid has no concrete consensus sequence required for cleavage. We sought to characterize what molecular features of the GDF8 cut site were required for tolloid processing. To this end, we identified several residues near the cut site that when mutated significantly reduced latent GDF8 activation by tolloid. Although GDF8 is tightly regulated through the formation of a latent procomplex, there are also extracellular antagonists that will bind to GDF8 and prevent it from signaling. Of the antagonists that target GDF8, the WFIKKN family is by far the most specific. However, the driver of this specificity was not well defined. Here, we characterized the follistatin domain (FSD) of WFIKKN2. Previously, the FSD had been identified as a primary driver of the high affinity interaction between GDF8 and WFIKKN. We solved the crystal structure of the WFIKKN2 FSD and identified key residues for antagonism and characterized how the FSD of WFIKKN contributes to antagonism. Together, our in vitro data provide valuable insight into the molecular mechanisms dictating GDF8 regulation in vivo and has the potential to be leveraged by the pharmaceutical industry to develop novel approaches for GDF8 inhibition to combat muscle wasting.

ATP in the tumor microenvironment is 1,000-10,000 times higher than in normal tissues and has recently been found to play many roles in tumorigenesis, including metastasis, which is associated with up to 90% of cancer related death. Metastasis starts with a process called epithelial to mesenchymal transition (EMT). Our lab has previously described that internalization of extracellular ATP (eATP) by cancer cells (in vitro and in vivo) by macropinocytosis in human cancer cells elevates intracellular ATP levels, and enhances cell proliferation and resistance to anticancer drugs. Most recently, we have reported ATP’s roles in inducing EMT and other early metastatic activities independent of TGF-beta in human lung cancer cells. Traditionally, TGF-beta has been considered to be important and necessary for EMT induction, but our published and more recent preliminary findings have prompted us to reconsider ATP as an alternative master inducer and regulator of EMT. In this study, we tested the hypothesis that ATP, like TGF-beta, is a master inducer and regulator of EMT, triggering EMT in a fashion similar to but non-identical to TGF-beta. Transwell assays revealed that addition of extracellular ATP (eATP) to human lung cancer cells induced both cell migration and invasion at higher rates than those of TGF-beta. Fluorescence microscopy exhibited that eATP treatment induced cell morphology changes indicative of EMT. ATP assays showed that eATP treatment resulted in significantly elevated intracellular ATP levels while TGF-beta treatment did not change the iATP level. This result indicates that although the treatments of eATP or TGF-beta all result in migration and invasion, they also lead to some different intracellular responses. The combined cell treatment with lower concentrations of eATP and TGF-beta led to some noticeable additive effect in invasion activity, suggesting that these two molecules may induce invasion by activating EMT through similar but non-identical signaling pathways. RNA sequencing (RNAseq) results reveal that, like TGF-beta, ATP induced expression of genes involved in EMT, with many Mesenchymal (M) genes upregulated and Epithelial (E) genes downregulated. This is consistent with ATP’s role as an EMT inducer and regulator. Differently from TGF-beta, ATP induced significant alteration of expression of fewer genes. It is noteworthy that eATP induced fewer significant gene upregulations (ATP : TGF-beta = ~2:3) and downregulations (ATP : TGF-beta = ~1:4) than TGF-beta at 6 hours treatment. The exact reasons for these reduced numbers are presently unclear. However, the multifunctional nature of ATP, especially its intracellular activities after internalization, is speculated to be involved in the reduction. The additional functions of ATP as a transcriptional cofactor, protein phosphorylation, and direct energy provision may be able to compensate for the fewer and lower gene expressions. Some of these additional functions of ATP may partially compensate for its transcriptional activity, so that EMT could still be achieved with fewer changes in and lower gene expression.

Numerous stresses on the heart, both physiological and pathological, present challenges to ER proteostasis in the heart. Simultaneously, various cardiac cell lineages respond to injury in ways requiring increased protein flux through the ER. While cardiac myocytes react to these stresses largely through hypertrophy or cell death mechanisms, other non-myocyte cells in the heart respond in more dynamic ways, including proliferation and differentiation. Additionally, virtually all cardiac cells secrete signaling cytokines or hormones both at baseline and during injury. Thus, precisely when ER proteostasis is most critical, cells are challenged with conditions which impair ER protein folding. ER unfolded protein response signaling attempts to respond to these challenges through the three main ER stress sensors, PERK, IRE1, and ATF6α. All sense protein misfolding and induce downstream signaling which, to varying degrees, includes global translational attenuation with concurrent upregulation of chaperones, protein disulfide isomerases, and ER-associated degradation machinery. Of these pathways, ATF6α is the most extensively characterized as adaptive in the heart and other tissues. Additionally, recent studies have identified multiple noncanonical ATF6α gene programs which are only indirectly related to ER proteostasis but are nevertheless critical to the ability of ATF6α to preserve tissue function. Research on the effect of ATF6α in the heart had previously been restricted to ventricular myocytes. Though these cells are the most direct effectors of cardiac function, this ignores critical roles played by other cardiac non-myocyte cells. This work examines the effects of ATF6α gain- and loss-of-function in three model systems designed to uncover previously unknown roles for ATF6α signaling in cardiac non-myocytes. ATF6α global knockout mice were found to progress to heart failure more quickly than wild-type counterparts following permanent-occlusion myocardial infarction, an unexplored disease model in the context of ATF6α signaling. We subsequently explored the role of ATF6α in two murine cardiac non-myocyte cell types. The first, c-Kit+ cardiac stem cells, required ATF6α both for survival and stemness while ATF6α loss-of-function induced multiple lineage markers. Second, we identified roles for ATF6α in limiting fibrosis in a pressure overload injury model and found that ATF6α blunted activation of isolated adult murine ventricular fibroblasts.

Intratumoral extracellular ATP (eATP), at levels of 100-700 μM or 103 to 104 times higher than in normal tissues, has been known to induce epithelial-mesenchymal-transition (EMT) of cancer cells via purinergic receptor signaling. However, the exact induction mechanisms are far from fully known. We previously described that eATP is internalized by cancer cells in vitro and in vivo by macropinocytosis in human non-small cell lung cancer A549 and other lung cancer cells, drastically elevates intracellular ATP levels, enhances cell proliferation and resistance to anticancer drugs. In this study, we tested the hypothesis that eATP and macropinocytosis-internalized eATP also induces EMT and other early steps of metastasis. Floating cells, fence assay, and Transwell assays were used to show that ATP induces cell detachment, new colony formation, migration and invasion in human A549 and other lung cancer cells. Western blots were used to detect ATP-induced changes in EMT-related proteins; Confocal microscopy was used to demonstrate ATP-induced metastasis-related cell morphological changes. Inhibitor and siRNA knockdowns were used to determine P2X7’s involvement in the ATP-induced EMT. CRISPR-Cas9 knockout of the SNX5 gene was used to identify macropinocytosis’ roles in EMT and cancer cell growth both in vitro and in vivo. Student’s t-test and one-way ANOVA were used to determine statistical significance, P

Osteocytes are the most numerous cells in bone, working from within the mineralized matrix to exert control over their local environment and cells on the bone surface. Healthy osteocytes are key to bone homeostasis, but the full functional importance of their local remodeling activity, called perilacunar/canalicular remodeling (PLR), is only recently being discovered. This work uses multiple mouse models to investigate the molecular mechanisms controlling osteocyte PLR and demonstrates that repression of PLR causes severe loss of bone material quality. Additionally, osteocyte dysfunction is shown to disrupt bone homeostasis during skeletal disease and to promote degradation of overlying cartilage in the joint. Finally, we begin to determine the extent to which PLR is mechanosensitive, revealing distinct site- and magnitude-specific modulation of osteocyte behavior with applied load. Together these findings reveal new essential roles for osteocytes and PLR within bone and joints during homeostasis and disease.

Nearly all life history traits scale with body size, imparting incredible importance on control of growth and size during animal development. Nutrition, genetic and environmental inputs influence the growth rate during development to determine final body size. These inputs are processed by the insulin/insulin-like growth factor signaling pathway (IIS), which is the major regulator of growth, and other pathways, like TGF-β, which modulate tissue growth or IIS. Mutations in the gene coding for the Drosophila TGF-β ligand Activinβ (Actβ) cause reduced final body size and accelerated growth termination. Using the Gal4/UAS system, I show Actβ is expressed in distinct cell types in the nervous system. Using rescue experiments, I show the Actβ phenotypes can be rescued by overexpression of Actβ in some, but not all, of the cell types in which it is endogenously expressed. Additionally, the growth rate of Actβ mutants is reduced, demonstrating the size phenotype is not simply due to early growth termination from precocious timing. Muscle-specific knockdown of the TGF-β signaling transducer/transcription factor dSmad2 also reduces body size, identifying muscle is a target tissue of the Actβ signal. The change in body size is due to a reduction in the size of skeletal muscles, not a systemic reduction in size. Autophagy markers are upregulated in Actβ mutants but, surprisingly, overexpression of autophagy regulators does not rescue the Actβ size phenotype, indicating Actβ regulation of autophagy is TOR-independent. These results provide new insights into mechanisms of body size and muscle size control during animal development. This thesis details the functions of Actβ in the regulation of body size and developmental timing. Chapter 2 describes the study of how Actβ controls body size and developmental timing. Chapter 3 investigates the roles of Actβ in regulating metabolism and IIS.

Congenital heart disease represents the most common form of birth defect and is present in up to 1% of all live births. Of particular interest to our group is the treatment of single ventricle disease, specifically hypoplastic left heart syndrome (HLHS) in which left-sided heart structures including the left ventricle, aorta, and mitral valve are malformed. We utilize tissue engineering principles to provide palliative treatment with Tissue-Engineered Vascular Grafts (TEVG) that allow for growth of the child’s own tissues. The TEVG overcomes limitations present in other composite grafts including thrombogenicity and lack of growth capacity. One limitation of our TEVG is that a small percentage of children develop critical stenosis requiring the utilization of interventional cardiovascular techniques. Developing both large and small animal models of TEVG stenosis will allow us to better understand clinical data sets as well as provide models to better characterize the development of neotissue formation and stenosis. The long-term goal of this project is to develop a second-generation composite vascular graft for use in children. The objective of this study is to elucidate the mechanism of TEVG neotissue formation in immunocompetent mice (Mus musculus) and characterize neotissue formation in a sheep (Ovis aries) model. Echocardiography, angiography, and magnetic resonance imaging are routinely used to evaluate children that have received the TEVG to treat HLHS. While angiography remains the clinical gold standard for evaluation of the lumen it only indirectly evaluates the vessel wall. Therefore, we sought to evaluate intravascular ultrasound (IVUS) to characterize neotissue formation in an in vivo ovine surgical model. IVUS demonstrated close correlation to angiographic and histologic changes. Additionally, IVUS measurement of graft lumen morphometry correlates with angiography measurements. This provides further data to support the use of IVUS in a clinical setting. Future preclinical experiments will utilize this modality as an additive approach to characterize the development of TEVG stenosis and neotissue remodeling over time, with specific regard to response to treatment with both interventional and therapeutic approaches.Previous research by our lab C57BL/6 mice has suggested that the transforming growth factor-ß (TGF-ß)/Smad2-3 pathway is involved in the development of TEVG stenosis. While the specific role of the TGF-ß pathway in the development of stenosis remains unknown, this pathway is upregulated in stenotic TEVG. To this end, this work seeks to evaluate the TGF-ß pathway in Cdh5-lineage cells in vivo in mice implanted with TEVG. A conditional genetic murine model was developed with endothelial-derived (Cdh5) loss of the TGF-ß receptors, TßRI and TßRII. Although TEVG have a low incidence of stenosis in both treated and control animals, Cdh5 TGF-ß pathway modulation results in altered neotissue formation including collagen production and polymeric degradation at early timepoints. We rationalize that by elucidating the cell(s) of origin for the neotissue and the role of TGF-ß receptor pathway will enable the development of targeted therapies in combination with TEVGs to improve clinical outcomes for children.

The transforming growth factor (TGF)-beta family of proteins drive normal embryonic development, while dysregulation of TGF-beta signaling contributes to developmental disorders, cancer progression and dissemination, and fibrosis. Signaling by TGF-beta initiates upon ligand binding to transmembrane receptor proteins, which then promote gene expression changes through phosphorylation and activation of intracellular Smad proteins. Functional activation of the TGF-beta receptors is carefully regulated through integration of post-translational modifications, spatial regulation at the cellular level, and receptor availability at the cell surface.While the majority of TGF-beta receptors reside intracellularly, they can be rapidly mobilized to the cell surface in response to Akt activation, thereby increasing the cell’s responsiveness to TGF-beta. Because TGF-beta is known to induce phosphorylation of Akt, I investigated whether TGF-beta regulates translocation of its own receptors to the cell surface and thus amplifies its own response. Through selective biotinylation of cell surface proteins, I found that TGF-beta induced a rapid increase of type I and type II TGF-beta receptors at the plasma membrane. This receptor upregulation was inhibited by blocking Akt phosphorylation, or in the presence of TGF-beta type I receptor kinase inhibitors. Furthermore, attenuation of the Akt-mediated increase in cell surface receptor presentation decreased Smad activation and TGF-beta-induced gene expression responses. I also observed that while TGF-beta treatment caused an overall increase in cell surface TGF-beta receptors, it also induced an increased rate of receptor endocytosis, suggesting an overall amplification of TGF-beta receptor cycling. Together, these data outline a novel response amplification mechanism, through a ligand-induced, rapid mobilization of cell surface receptors from intracellular stores.Furthermore, BMP-4 caused cells to upregulate TGF-beta receptors, leading to subsequent autocrine activation of TGF-beta-responsive Smads and gene expression. Inhibition of Akt phosphorylation or inhibition of TGF-beta type I receptor kinase attenuated the autocrine TGF-beta signaling activation in response to BMP. The balance between BMP and TGF-beta signaling pathways is essential for proper spatial and temporal regulation of tissue specification during development, and has been observed biochemically, although the underlying mechanisms are not clear. This work provides context for activation of TGF-beta signaling by BMP, presenting a means by which cells could gain sensitivity to TGF-beta in the presence of BMP.

Volume overload (VO) induced heart failure results from an increase in blood volume (preload) to the heart. The heart responds to increases in hemodynamic load through compensative remodeling. VO has a distinct pattern of remodeling compared to pressure overload induced heart failure, which results in fibrosis. VO results in a net decrease in extracellular matrix (ECM). This loss of ECM contributes to the progression of the disease due to the loss of structural integrity. Since cardiac fibroblasts (CFs) are the main cells responsible for maintaining ECM in the heart, we characterized the in vitro phenotype of CFs isolated from a rat VO model, aortocaval fistula (ACF). Compared to sham operated animals, ACF fibroblasts displayed a phenotype that we described as “hypofibrotic”. ACF CFs secreted relatively less collagen and profibrotic molecules, such as a-smooth muscle actin (aSMA) and connective tissue growth factor (CTGF). Interestingly, ACFs produce approximately twice as much transforming growth factor-ß1 (TGF-ß), a key profibrotic stimulus, as their sham counterparts. However, there were no changes in the canonical TGF-ß pathway that could account for the hypofibrotic phenotype observed in ACF fibroblasts. Since others have shown that the cytoskeleton and the Rho/ROCK pathway play a role in fibroblast phenotype, we characterized the actin cytoskeleton in sham and ACF fibroblasts. We found that ACF CFs have significantly less F-actin than sham CFs. We were able to show that it is possible the actin cytoskeleton might account for phenotypic differences in CFs by chemically altering the amounts of F-actin and G-actin. When the cells were treated with a ROCK inhibitor, which allows F-actin to depolymerize into G-actin, CFs displayed a more hypofibrotic phenotype. Conversely, enhancement of F-actin with jasplakinolide treatment forced the CFs to have a profibrotic phenotype. Numerous studies have linked substrate modulus with effects on the cytoskeleton. Stiff substrates tend to increase cytoskeletal organization and increase F-actin resulting in stress fiber formation in vitro. We wondered if changes in tissue modulus may account for differences in observed phenotype. To know if changes in tissue modulus account for changes in CF phenotype, we first characterized the tissue stiffness changes. We used biaxial tensile testing, which yields a direct measure of tissue modulus, an intrinsic property of the material. Here we show that ACF rats have approximately half the tissue modulus compared to control rats. Since increased stiffness is positively correlated with CF changes towards a more profibrotic phenotype, we postulated that decreased substrate stiffness may lead to a more hypofibrotic phenotype. Specifically, we hypothesized that sham CFs on softer substrates would have a more hypo-fibrotic phenotype; conversely, CFs from ACF would behave more like normal cells on a higher stiffness. Although the phenotype of sham CFs was shifted to a more hypofibrotic direction on soft substrates, ACF fibroblasts had many indications of a dampened response to stiffness reminiscent of the effect known as “mechanical memory” described by others 1,2.

Transforming Growth Factor – beta signaling plays important, pleiotropic roles in embryonic development as well as tissue homeostasis. TGF-beta signaling dose is tightly regulated in a context-specific manner and even subtle perturbations result in a spectrum of disease phenotypes of varying severity. In the canonical TGF-beta pathway, TGF-beta signals are mediated by activated receptor-regulated Smads (R-Smads, Smad2/3) that form a complex with Smad4 and participate in transcriptional regulation of TGF-beta-mediated genes with sequence specific DNA binding factors and context-specific co-regulators. Both Smad2/3 and Smad4 regulate signal duration and intensity differentially. This is consistent with the variability and spectrum of phenotypes observed upon their conditional deletions in mice, with Smad2/3-associated phenotypes being more severe than those associated with Smad4. However, precise mechanisms that modulate the functional and phenotypic differences attributed to Smad2/3 and Smad4 are poorly understood. Tripartite motif containing (Trim) 33 - a co-regulator of both activated Smad2/3 and Smad4, is a candidate gene for understanding fine tuning mechanisms of TGF-beta signaling. Here, I have investigated the role of Trim33 in early embryogenesis. My results show that Trim33 controls visceral endoderm differentiation in vitro and pre-cardiac mesoderm differentiation in vivo. Epiblast-specific Trim33 mutants die during the second half of gestation as a result of ensuing cardiac failure. My findings imply that Trim33 is required for appropriate early mesendoderm differentiation soon after its induction, regulates TGF-beta superfamily signaling in a biphasic manner in the pre-cardiac mesoderm, and that in more committed cell lineages Trim33 is dispensable.

Regulatory T cells (Tregs) are critical for the maintenance of immune homeostasis and prevention of autoimmunity. In the non-obese diabetic (NOD) mouse model of type 1 diabetes, Tregs accumulate in the inflamed islets of langerhans and delay or prevent the destruction of insulin-producing beta (β) cells. The regulators of Treg homeostasis and function are yet to be completely understood in the context of chronic tissue inflammation. In this work we focused on the specificity of Tregs for their cognate antigen, the dynamics of these Tregs, and influence of the cytokines IL2 and TGFβ. This was accomplished by examining the target tissue compared with peripheral lymphoid sites for different aspects of Treg biology using flow cytometry, histological analysis, and TCR sequencing. We found that islet Tregs were potent mediators of disease prevention, had most recently and strongly been exposed to their cognate antigen, and we could identify a highly activated, antigen specific subset using cell surface markers CD103, ICOS, and TIGIT. Islet Tregs were a dynamic population constantly turning over, but these Tregs were not circulating through the LNs and instead isolated to the tissue. They were specific for known autoantigens, such as insulin, but the highly activated, antigen reactive subset of Tregs were also clonally expanded due to reactivity with unknown islet antigens. After adoptive transfer, both CD103+ and CD103- Tregs prevented diabetes development and were more potent than Tregs from peripheral lymphoid organs. Their efficacy could be increased with a short treatment with IL2. When Tregs cannot respond to TGFβ signaling, their function in preventing diabetes increases, while their ability to prevent autoimmune neuropathy decreases. Overall, our findings that islet Tregs are highly localized, potent effector Tregs that can be manipulated with cytokines implicate the importance of antigen specific tissue Tregs in preventing diabetes development and the impact of Treg cytokine signaling on disease pathogenesis.

Antecedentes Las cicatrices hipertróficas suponen una de las complicaciones más relevantes en el campo de la cirugía debido a sus implicaciones cosméticas y funcionales. Transforming Growth Factor-beta (TGF-β) es uno de los reguladores más representativos responsables de una cicatrización anómala. En estudios previos donde se han utilizando inhibidores de la acción del TGF-β1 en enfermedades caracterizadas por exceso de fibrosis han demostrado efectos prometedores. El objetivo del presente estudio es analizar el efecto de la administración tópica del péptido inhibidor del receptor tipo III del TGF-β1 (P144®) sobre cicatrices humanas implantadas en ratones atímicos.Material y métodos Se tomaron muestras de cicatrices hipertróficas de 30 pacientes y se implantaron en 60 ratones atímicos. Los ratones se dividieron en dos grupos, grupo A (placebo) y grupo B (tratamiento). El grupo C (basal) fue constituido por las muestras de las cicatrices hipertróficas sin implantar. Una vez que prendieron todas las cicatrices sobre los ratones se procedió a la aplicación diaria de un lipogel que contenía o bien un vehículo o bien P144 sobre todas las muestras durante 15 días consecutivos. Después de finalizado el tratamiento, los animales fueron sacrificados y se extrajeron dichas cicatrices hipertróficas. El área total, el grosor y el área ocupada por fibras de colágeno fueron cuantificados para su comparación entre los distintos grupos. El estudio inmunohistoquímico se realizó para determinar la expresión de colágeno I, colágeno III y fibrilina-1. Resultados El 83.3% de los xenoinjertos prendieron totalmente. El tiempo medio para el prendimiento fue de 35±5.4 días. Diferencias estadísticamente significativas se encontraron en el área total, grosor de la cicatriz y área ocupada por fibras de colágeno en la comparación entre los tres grupos. La reducción en el área total secundario al tratamiento fue de un 10% en comparación con el grupo placebo. Un aumento significativo en la expresión de fibrillin-1 y una disminución de colágeno I fue hallado en el grupo que recibió el tratamiento comparado con el grupo basal. Conclusiones La aplicación tópica del péptido inhibidor del TGF-β1 en el modelo “in vivo” de cicatrices hipertróficas implantadas en ratones atímicos ha demostrado revertir la cicatrización a su curso normal con la consiguiente disminución del contenido en colágeno y reordenamiento de las fibras elásticas. No cabe duda de que existe un progresivo conocimiento en la fisiopatología de la cicatrización hipertrófica, sin embargo aún sigue siendo necesario la búsqueda de un modelo animal que permita estudiar este desorden de forma global. Con ello, alcanzaremos un conocimiento aún más extenso que facilitara nuevos tratamientos enfocados en bloquear las vías reguladoras presentes en estos desórdenes fibróticos.

Protein translational regulation by RNA binding proteins (RBPs) is a critical process in maintaining homeostasis. Epithelial to mesenchymal transition (EMT) is a process in which epithelial cells de-differentiate and become mesenchymal, increasing the propensity toward tumorigenesis and/or metastasis. We have identified a heterogeneous nuclear riboprotein E1 (hnRNP E1)-mediated post-transcriptional operon that controls transcript-selective translational regulation of epithelial / mesenchymal transition (EMT)-associated genes. In this regulatory mechanism, hnRNPE1 binds to the 3’-UTR of select transcripts and silences their translation. TGFß reverses translational silencing through Akt2-dependent phosphorylation of hnRNP E1 at Ser-43, resulting in loss of hnRNP E1 binding to RNA. We have identified approximately forty pro-EMT / metastatic mRNAs that are regulated by this hnRNP E1 operon and our preliminary studies have revealed a short stretch of nucleic acids, that we have termed the BAT element (TGF-beta activated translational (BAT) element), present in their respective 3’-UTRs that may be responsible for hnRNP E1 binding. Herein, through the use of in-vitro and in-vivo assays, we demonstrate the contribution of BAT element mutations and constitutively high levels of pSer43 hnRNP E1 to cancer tumorigenesis and metastasis.

Thyroid cancer is the most common endocrine cancer and its incidence rates continue to rise. Papillary thyroid cancer (PTC) is the most common type and it is generally treatable with surgery followed by radioactive iodine (RAI) therapy. Thyrotropin (TSH)-stimulated, Sodium/Iodide Symporter (NIS)-mediated RAI uptake (RAIU) is the basis for RAI therapy. While most patients show good prognosis, a subset of patients with advanced thyroid cancer do not benefit from RAI therapy due to reduced NIS expression/function. The objective of this study was to investigate the molecular mechanisms of NIS modulation and to identify novel reagents that selectively increase RAIU in thyroid cells, such that the subset of patients could benefit from RAI therapy. Most common genetic alterations in PTCs are the BRAF(V600E) mutation that activates MEK/ERK signaling and RET/PTC rearrangement that activates both PI3K/Akt/mTOR and Ras/BRAF/MEK/ERK pathways. Hsp90 is a chaperone to many of these oncoproteins. TGF-beta is a cytokine in invasive fronts of thyroid tumors, which also activates MEK/ERK. These signaling nodes not only contribute to tumor progression but also result in reduced NIS expression/function. Here we report that among cell culture-grade small molecule inhibitors tested, Akt inhibitor (Akti1/2) increased TSH-stimulated RAIU to the greatest extent in PCCl3 rat thyroid cells, when compared to inhibitors of MEK, PI3K or Hsp90. We identified Apigenin, a plant-derived flavonoid that further increased RAIU by increasing iodide influx rate in combination with Akti1/2 and this required p38 MAPK activity, but not PKC-delta. The effect was confirmed in BRAF(V600E) or RET/PTC1-expressing PCCl3 cells, and in primary thyroid tumor cells from TR-beta(PV/PV) mice.We then took advantage of small molecule inhibitors targeting Akt, MEK, PI3K, Hsp90 or BRAF used in clinical trials for chemotherapy of solid tumors. As their toxicity profiles are well-accepted, if they increase thyroidal RAIU, they could be immediately employed in clinical trials to improve RAI treatment efficacy. We identified that PI3K inhibitor GDC-0941 outperformed other inhibitors in increasing TSH-stimulated RAIU in PCCl3 and BRAF(V600E)-expressing PCCl3 cells, by greatly decreasing iodide efflux rate. Alarmingly, TGF-beta dramatically reduced RAIU even upon treatment with inhibitors. Apigenin counteracted the RAIU reduction by TGF-beta by increasing NIS protein. Taken together, co-treatment of GDC-0941 with Apigenin may increase therapeutic efficacy of RAI in invasive fronts of thyroid cancer. While much is known about signaling nodes, little is known about microRNAs (miRs) regulating NIS and RAIU. We identified that miR-339-5p decreased endogenous NIS mRNA and RAIU in MCF-7 human breast cancer cells and in PCCl3 cells. Among 38 miRs deregulated by TGF-beta, Akti-1/2 or 17-AAG in PCCl3 cells, 18 were conserved in humans. One of the 18 miRs, miR-195, was predicted to bind to hNIS-3’UTR and its overexpression decreased hNIS-mediated RAIU in MCF-7 cells. MiR-339-5p was modestly increased, yet miR-195 was significantly decreased in PTCs. Expression profiles of the 18 miRs were able to separate most PTCs from non-malignant thyroid tissues. Taken together, we hope that these studies would serve as valuable tools to develop novel strategies for optimal use of RAI in clinical management of thyroid cancer.

The formation of the palate (palatogenesis) is a complex developmental process that when disrupted results in cleft palate, a common human birth defect, occurring in 1 out of every 2000 babies. The palate is formed from a pair of palatal shelves composed of a mesenchymal core covered by a layer of surface epithelium. Additionally, prefusion palatal shelves are covered by a thin layer of transient peridermal cells. The shelves grow downwards along each side of the tongue, elevate and fuse forming the midline epithelial seam (MES), which is subsequently removed to form a fused palate. The TGF-β superfamily plays an essential role in MES removal as well as in craniofacial growth and patterning. Here we examine the mechanism of Smad-dependent (canonical) and Smad-independent (non-canonical) signaling in the palatal mesenchyme and epithelium, and the regulation of Tgfb3 palatal expression.

Cardiovascular disease is now the leading cause of death worldwide. Serum amyloid A (SAA), a positive acute phase reactant, along with C-reactive protein is used clinically as a marker of cardiovascular disease risk. However, recent data has shed light on a possible causal role of SAA in the development of atherosclerosis, the most pervasive form of cardiovascular disease. Several inflammatory diseases such as diabetes and obesity are known to confer increased risk of developing cardiovascular disease. Individuals with these diseases all have modest but persistent elevation of SAA. To determine if SAA caused the development of atherosclerosis, apoe-/-chow fed mice were injected with either an adenoviral vector expressing human SAA1 (ad-hSAA1), a null adenoviral vector (ad-Null) or saline. Human SAA levels rapidly increased, albeit briefly then returned to baseline within 14 days in mice that received ad-hSAA1. After 16 weeks, mice that received ad-hSAA1 had significantly increased atherosclerosis compared to controls on the aortic intimal surface (pldlr-/- mouse on a C57BL/6 background and fed a pro-atherogenic western diet for 12 weeks. There was a significant increase in atherosclerotic lesion area on the aortic intimal surface (p

The identification of beige fat within the last decade, its ability to burn energy in adult humans, and its great potential for therapeutic applications has motivated many to work towards understanding how beige fat is made. Although several proteins have been identified as important for beige fat differentiation, it is clear that the differences that distinguish beige fat from other types of fat cannot be explained by the presence of these proteins alone. Additional regulatory proteins, including transcription factors and their co-factor proteins, must be involved.I took advantage of two important developments in the field of fat differentiation to develop two high throughput approaches that identified new transcription factors involved in beige fat: (1) our ability to culture beige fat cells by differentiating white fat pre-adipocytes in the presence of Rosiglitazone and (2) the established role for PRDM16 as required for beige fat differentiation. In brief, I combined RNA-seq data from beige fat cells and proteomics data from Rosiglitazone-dependent PRDM16 protein complexes to identify a set of candidate transcription factors involved in beige fat differentiation. The most promising candidate among this pool of putative beige fat regulatory transcription factors was GTF2IRD1.I determined that GTF2IRD1 is a PRDM16-interacting transcription factor that is enriched in beige and brown fat cells. In vivo, GTF2IRD1 is enriched in brown adipose tissue and is increased in beige and brown fat in response to beta-3-adrenergic stimulus. In the presence of the potent PPARgamma agonist Rosiglitazone, GTF2IRD1 overexpression enhances and shRNA-mediated knockdown reduces beige fat differentiation. GTF2IRD1 represses TGF-beta-mediated inhibition of beige fat differentiation. In summary, my data strongly supports that GTF2IRD1 is an essential regulator of beige fat differentiation through interaction with PRDM16 and inhibition of TGF-beta-mediated repression of differentiation.

Immune molecules, including complement proteins, C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement proteins localize to synapses and refine the developing retinogeniculate system via C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. RGCs grown in the presence of astrocytes significantly upregulated C1q compared to controls, implicating an astrocyte-derived factor in neuronal C1q expression. A major goal of my dissertation research was to identify the signals that regulate C1q expression and function in the developing visual system. In this study, I have identified transforming growth factor beta \((TGF-\beta)\), an astrocyte-secreted cytokine, as both necessary and sufficient for C1q expression in RGCs through an activity-dependent mechanism. Specific disruption of retinal \(TGF-\beta\) signaling resulted in a significant reduction in the deposition of C1q and downstream C3 at retinogeniculate synapses and significant synaptic refinement defects in the retinogeniculate system. Microglia engulfment of RGC inputs in the lateral geniculate nucleus (LGN) was also significantly reduced in retinal \(TGF\beta\)RII KOs, phenocopying the engulfment defects observed in C1q KOs, C3 KOs, and CR3 KOs. Interestingly, in C1q KOs and retinal \(TGF\beta\)RII KOs, microglia also failed to preferentially engulf less active inputs when retinal activity was manipulated, suggesting that retinal activity and \(TGF-\beta\) signaling cooperatively regulate complement mediated synaptic refinement. In support of this hypothesis, blocking spontaneous activity in RGC cultures significantly reduced C1q upregulation by \(TGF-\beta\). Moreover, manipulating spontaneous retinal activity in vivo modulated C1q expression levels in RGCs and C1q deposition in the LGN. Together these findings support a model in which retinal activity and \(TGF-\beta\) signaling control expression and local release of C1q in the LGN to regulate microglia-mediated, complement-dependent synaptic pruning. These results provide mechanistic insight into synaptic refinement and, potentially, pathological synapse loss which occurs in the early stages of neurodegenerative diseases concurrently with aberrant complement expression and reactive gliosis.

Breast cancer is the second leading cause of cancer death in women in the United States. Metastasis accounts for the death of ~90% of these patients, yet the mechanisms underlying this event remain poorly defined. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that functions to suppress tumorigenesis in mammary epithelial cells (MECs). Interestingly, mammary tumorigenesis converts TGF-beta from a tumor suppressor to a tumor promoter through molecular mechanisms that remain incompletely understood. Changes in tissue compliance promote the acquisition of malignant phenotypes in MECs in part through the activity of the extracellular crosslinking enzyme, lysyl oxidase (LOX), which regulates desmoplastic reactions and metastasis. We show that TGF-beta induces the synthesis and secretion of LOX from normal and malignant MECs, and in breast cancers produced in mice. Additionally, antagonizing LOX activity reduces TGF-beta-mediated invasion and epithelial-mesenchymal transition (EMT) in breast cancer cells. We further show that increased extracellular matrix (ECM) rigidity promotes the proliferation of malignant MECs, a cellular reaction that is abrogated by inhibiting TGF-beta signaling or by antagonizing LOX activity. As a possible mechanism for the influence of matrix rigidity on TGF-beta signaling, we sought to define the microRNA expression profiles induced by TGF-beta and ECM rigidity during metastatic progression. Through global profiling analyses, we identified microRNAs whose expression was induced by TGF-beta and associated with metastatic potential. In doing so, we identified miR-181a as a TGF-beta-regulated “metastamir” that enhanced the metastatic potential of breast cancers by promoting their acquisition of EMT, migratory, and invasive phenotypes. Mechanistically, inactivating miR-181a elevated the expression of the pro-apoptotic molecule, Bim, which sensitized metastatic cells to anoikis. Along these lines, miR-181a activity was essential in driving pulmonary micrometastatic outgrowth and enhancing the lethality of late-stage mammary tumors in mice. Finally, miR-181a expression is dramatically and selectively upregulated in metastatic breast tumors, particularly triple-negative breast cancers, and is highly predictive for decreased overall survival in human breast cancer patients. Collectively, our findings implicate LOX and miR-181a expression as novel diagnostic markers for metastatic progression, as well as an innovative therapeutic targets to treat metastatic breast cancers.

Myopia (near-sightedness) is one of the most common ocular disorders in humans. Due to the dramatic increases in prevalence of myopia worldwide, especially in children and young adults, myopia has also become a significant public health problem, both socially and economically. While the prevalence and severity of myopia continue to increase, effective therapeutic interventions for myopia remain limited. Currently, management of myopia is largely limited to traditional optical corrections - spectacles, contact lenses, and refractive surgery - which correct distance vision but have no effect on myopia progression. While slowed myopia progression has been reported in clinical studies using the contact lens-based, corneal reshaping therapy and atropine, a pharmaceutical agent, these approaches come with limitations and in the latter case, significant ocular side-effects. Uncontrolled progression may lead to high "degenerative" myopia, for which posterior scleral reinforcement surgery remains the only treatment option and a last resort directed at preserving vision. Thus there is a clear need for new myopia control treatments. Understanding more about the molecular and cellular mechanisms underlying myopic eye growth has the potential to uncover novel treatment options. This dissertation presents results from three investigations into the role of the retinal pigment epithelium (RPE) in eye growth regulation, focusing on molecular and cellular mechanisms, and using both in vivo animal models and in vitro cell culture models. In the first in vivo study (Chapter 2), we investigated expression of candidate genes in chick RPE of imposing short-term optical defocus. Specifically, gene expression levels of the three bone morphogenetic proteins (BMP-2, 4 & 7) were examined after 2 and 48 h of treatment, with negative and positive lenses used to impose defocus of opposite sign. These growth factors were observed to be differentially and bidirectionally expressed in RPE, expression generally increasing with imposed myopia, which is associated with ocular growth inhibition. Because eyes had little chance to change their dimensions with such short-term lens treatments, these genes are assumed to play important roles in the onset and early phase of defocus-induced ocular growth changes. For this reason, these genes represent potential targets for molecular-based myopia treatments. In the second study (Chapter 3), high-through gene expression profiling was employed to examine changes in gene expression in chick RPE with long-term imposed hyperopic defocus, which resulted in eyes being longer than normal and highly myopic. This DNA microarray screening revealed changes in the expression of many genes, including BMPs, noggin (NOG), dopamine receptor D4 (DRD4), retinoic acid receptor, beta (RARB), and retinal pigment epithelium-derived rhodopsin homolog (RRH). Some of these genes showed increased expression while others showed decreased expression. It is plausible that some may be linked the ocular pathological complications seen in myopia, while others may be linked to ocular growth regulation, the imposed visual conditions resulting in sustained, increased ocular growth. The third and final study (Chapter 4), addressed the possibility of an RPE site for the anti-myopia action of apomorphine (APO), a dopamine receptor agonist, observed in animal studies. We further investigated the possibility that TGF-â secretion from RPE mediates this inhibitory growth effect. APO applied to cultured human fetal RPE cells was found to alter the secretion of both TGFâ1 and TGFâ2, which was biased towards the basal (choroidal) side. These growth factors also exhibited constitutive polarized secretion, albeit biased in the opposite direction to APO-induced paracrine secretion. The results for APO are consistent with its observed inhibitory (anti-myopia) effects in vivo and offer the RPE as a possible site of action. In summary, the research reported in this dissertation provides evidence that RPE plays an important role in postnatal eye growth regulation, (including myopic growth), as a conduit for relaying growth modulatory retinal signals to choroid/sclera. Genes and molecules identified in these studies offer potential directions for novel anti-myopia treatments, with the RPE being a potential target for the same.

Cellular fate, once established, is usually stable for the lifetime of the cell. However, the mechanisms that restrict the developmental potential of differentiated cells are in principle reversible, as demonstrated by the success of animal cloning from a somatic genome through somatic cell nuclear transfer (SCNT). An increased understanding of the molecular determinants of cell fate has also enabled the reprogramming of cell fate using defined transcription factors; recently, these efforts have culminated in the discovery of four genes that convert somatic cells into induced pluripotent stem cells (iPSCs), which resemble embryonic stem cells (ESCs) and can give rise to all the cell types in the body. As a first step toward generating clinically useful iPSCs, we identified a small molecule, RepSox, that potently and simultaneously replaces two of the four exogenous reprogramming factors, Sox2 and cMyc. This activity was mediated by the inhibition of the Transforming Growth Factor-\(\beta\) \((Tgf-\beta)\) signaling pathway in incompletely reprogrammed intermediate cells. By isolating these stable intermediates, we showed that RepSox acts on them to rapidly upregulate the endogenous pluripotency factor, Nanog, allowing full reprogramming to pluripotency in the absence of Sox2. We also explored lineage conversion as an alternative approach for producing a target cell type in a patient-specific manner, without first generating iPSCs. A combination of pro-neural as well as motor neuron-selective factors could convert fibroblasts directly into spinal motor neurons, the cells that control all voluntary movement. The induced motor neurons (iMNs) displayed molecular and functional characteristics of bona fide motor neurons, actuating muscle contraction in vitro and even engrafting in the developing chick spinal cord when transplanted. Importantly, functional iMNs could be produced from fibroblasts of adult patients with the fatal motor neuron disease, amyotrophic lateral sclerosis (ALS). Given the therapeutic value of generating patient-specific cell types on demand, defined-factor reprogramming is likely to serve as an important tool in regenerative medicine. It is hoped that the different approaches presented here can complement existing technologies to facilitate the study and treatment of intractable human disorders.

Vertebrate limb morphogenesis is coordinated by a complex network of signaling centers that influence growth, differentiation and movement of the cells in the developing limb field. Several members of signaling molecules of the Transforming Growth Factor beta (TGF-beta) family are shown to be potent regulators of limb morphogenesis. Smad4 is a central mediator of the TGF-beta-related signaling in a variety of developmental contexts. Zygotic inactivation of Smad4 results in lethality at peri-gastrulation stage. We have generated a conditional knockout of Smad4 that disrupted TGF-beta-related signaling in the developing limb ectoderm. Ectodermal disruption of Smad4 signaling leads to severe patterning defects in autopods and in some cases, zeugopods with loss of posterior elements in forelimbs. However less severe patterning defects such as syndactlyly and ectopic nail growth occur in the hindlimbs as revealed by the skeletal analyses of the mutant limbs. These patterning defects are posteriorally biased and can be correlated to earlier defects in the shape of the developing limb bud beginning at embryonic day 10.5. This shape change was monitored over a 16 somite stage range (E10.5-11.5, 31ss-46ss) with the use of a ROSA reporter strain. Taken together the morphometric analyses of our mutant phenotype have shown a novel role for SMAD4 signaling in the development of the embryonic vertebrate limb.

Neurofibromatosis type 1 is a common autosomal dominant disorder affecting 1in 3500 individuals worldwide. Neurofibromin, the protein mutated in NF1 disease, is a GTPase activating protein (GAP) for Ras proteins, inactivating the Ras proteins H-Ras, N-Ras, K-Ras, M-Ras, R-Ras, and TC21. Missense mutations in the GAP related domain of neurofibromin cause NF1 disease, indicating that increased Ras activity is likely critical for disease pathogenesis. Loss of NF1 in Schwann cells causes formation of benign tumors known as neurofibromas. These tumors can become malignant forming malignant peripheral nerve sheath tumors (MPNSTs), which are a major source of morbidity for NF1 patients. TC21 is a member of the R-Ras family of small Ras GTPases. TC21 is an oncogene able to transform epithelial and fibroblast cell lines, and it is also capable of inducing tumors in vivo. PI3K is the predominant effector of TC21 transforming activity, and studies support the idea that not all features of NF1 mutant cells can be ascribed to the activation of the classical Ras proteins (e.g. H-, N-, K-Ras). We hypothesize that the effects of neurofibromin mutation that are unrelated to classical Ras-GTP may be explained by activation of the non-classical Ras protein TC21. The signaling pathway of TC21 is unclear. We activated all Ras proteins in vivo by deletion of Nf1 and using these mice along with mice deficient for TC21-/-, we examined the role and function of TC21 in development and tumorigenesis. Additionally, we used NF1-/- human MPNST cell lines with an acute loss of TC21 by shRNA to examine tumor growth in xenograft mice. We found that TC21 loss delayed benign neurofibroma formation in Nf1fl/fl;DhhCre mice. Nf1 loss increased mRNA encoding the cytokine transforming growth factor-beta (TGF-beta) and rendered Schwann cell progenitors insensitive to TGF-beta; these phenotypes could be rescued by TC21 and were mediated through TGF-beta receptors. Conversely, growth of Nf1;Trp53 brain tumors and NF1-/- MPNST sarcomas were accelerated by TC21 loss. MPNST from Nf1;Trp53 mice and NF1-/- MPNST xenografts had increased levels of TGF-beta mRNA and protein and blocking TGF-beta decreased sarcoma size induced by shTC21. The results are important because TGF-beta acts as a tumor suppressor in numerous types of benign tumors and is also known for its oncogenic role in cellular transformation and tumor progression. Our data elucidates TGF-beta as a therapeutic target in NF1 malignancy.

The TGF-beta superfamily, including TGF-betas and BMPs, is critical for normal embryonic development, as well as disease progression, and is tightly regulated both within and out of cells. In vitro, TGF-beta signaling mediated epithelial-mesenchymal transition (EMT) by activating mesenchymal genes and suppressing epithelial markers. We discovered that Wnt11 was directly regulated by the mediators of TGF-beta signaling, Smad proteins. The induction of Wnt11 expression was critical for TGF-beta associated activation of mesenchymal marker genes. Instead of modulating Smad proteins or activating canonical/beta-Catenin signaling, Wnt11 controlled mesenchymal gene activation through JNK signaling. Our findings, for the first time, demonstrated the cooperativity among the TGF-beta, Wnt11 and JNK signaling pathways in the context of EMT. Both TGF-beta and BMP signaling are involved in renal fibrosis, but with opposite functions. TGF-beta is a well known pro-fibrogenic factor, while BMP counteracts TGF-beta to protect kidney from injuries. In our study, transgenic expression of kielin/chordin-like protein (KCP), an inhibitor of TGF-beta and enhancer of BMP7, in renal epithelia attenuated the upregulation of mesenchymal genes in the injured kidney of unilateral ureteral obstruction (UUO) mouse model. These data demonstrated the importance of the balance of TGF-beta and BMP signaling in the progression of renal fibrosis and provided a new potential therapeutic target. During kidney development, both BMP7 and Tle4, a common corepressor, are present in metanepheric mesenchymal cells. However, their relationship is unknown. Here, we found that overexpression of Tle4 not only activated a BMP reporter, but also enhanced and sustained the upregulation of endogenous Id1 gene induced by BMP7. The effect of Tle4 on BMP signaling was through mediating Smad7 protein, for Tle4 repressed Smad7 expression and overexpression of Smad7 totally abolished the activation of the BMP reporter by Tle4. Our study provides a new potential mechanism for the regulation of BMP signaling in the kidney development.

Endochondral ossification involves a highly coordinated program of chondrocyte differentiation, proliferation, maturation, and hypertrophy. The bone morphogenetic protein (BMP) and transforming growth factor beta (TGFβ)/activin pathways are important regulators of these processes. The importance of extracellular antagonists as regulators of the duration, intensity and extent of BMP and TGFβ/activin signaling has been defined. For example, mice lacking the BMP receptor antagonist Noggin exhibit cartilage overgrowth concurrent with excessBMP activity. However, very little is known about the roles of intracellular inhibitors of BMP and TGFβ/activin pathways, such as the inhibitory Smad (I-Smad) proteins, Smad6 and Smad7. In vitro studies reveal that I-Smads 6 and 7 can regulate BMP- and TGFβ-mediated effects on chondrocytes. Although in vivo studies in which I-Smads were overexpressed in cartilage have shown that I-Smads have the potential to limit BMP signaling in vivo, the physiological relevance of I-Smad activity in skeletal tissues remains unknown. Furthermore, whether I-Smads impact TGFβ signaling in cartilage during development is unclear.This thesis includes two reviews (both have been published) and two original articles (one of which has been published). First, the known mechanisms by which BMP signaling regulate chondrogenesis, osteogenesis, and adipogenesis will be highlighted Chapter One, "BMP signaling in Skeletogenesis." Then, the roles of Smad proteins, which are the mediators of the canonical BMP/TGFβ pathways, in regulating skeletal development will be highlighted in Chapter Two, "Smad signaling in skeletal development and regeneration." Finally, the roles of Smad6 and Smad7 in endochondral bone formation will be described in Chapters Three ("Smad6 is essential to limit BMP signaling in cartilage development") and Four ("Smad7 regulates terminal maturation of chondrocytes during cartilage development in mice"). The last chapter will discuss the overall conclusions and future directions of my thesis project.

Myostatin is a member of the transforming growth factor-β (TGF-β) family and a strong negative regulator of muscle growth. As such, myostatin is an attractive therapeutic target for the treatment of muscle-wasting diseases such as muscular dystrophy. Here, we present crystal structures of myostatin in complex with two antagonist molecules: follistatin (Fst) and Fst-like 3 (Fstl3). These represent the first and only published structures of myostatin. Furthermore, these are the only structures of Fst-type molecules in complex with a ligand other than activin A. Both Fst and Fstl3 are staunch inhibitors of myostatin, and Fstl3 is in fact the only Fst-type molecule that has been identified in the serum bound to myostatin. We find that the prehelix region of myostatin very closely resembles that of TGF-β class members and that this region alone can be swapped into activin A to confer signaling through the noncanonical type I receptor ALK5. Furthermore, the N-terminal domain (ND) of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. Additionally, a unique continuous electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. The ND of Fstl3 also interacts uniquely with myostatin as compared to activin A, as it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. The Fstl3 ND also exhibits less plasticity than that of Fst, and select point mutations in it are detrimental to Fstl3 function. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand:Fst-type protein complexes. Altogether, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.

Programmed cell death (PCD) is an essential feature during the development of the central nervous system in Drosophila as well as in mammals. During metamorphosis, a group of peptidergic neurons (vCrz) are eliminated from the larval central nervous system (CNS) via PCD within 6-7 h after puparium formation. To better understand this process, we first characterized the development of the vCrz neurons including their lineages and birth windows using the MARCM (Mosaic Analysis with a Repressible Cell Marker) assay. Further genetic and MARCM analyses showed that not only Myoglianin (Myo) and its type I receptor Baboon is required for neuron cell death, but also this death signal is extensively regulated by endocytic trafficking in Drosophila melanogaster. We found that clathrin-mediated membrane receptor internalization and subsequent endocytic events involved in Rab5-dependent early endosome and Rab11-dependent recycling endosome differentially participate in TGF-β [beta] signaling. Two early endosome-enriched proteins, SARA and Hrs, are found to act as a cytosolic retention factor of Smad2, indicating that endocytosis mediates TGF-β [beta] signaling through regulating the dissociation of Smad2 and its cytosolic retention factor.

Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS) that is perpetuated by myelin-specific autoreactive CD4+ T cells. Interestingly, both healthy individuals and MS patients have myelin-specific T cells; however, these cells are of an effector-memory phenotype in MS patients, indicating previous exposure to antigen (Ag). Additional phenotypic analysis has demonstrated these cells are primarily of an effector-memory T helper (Th) 1 and Th17 lineage in MS patients. After activation in the periphery, myelin-specific T cells traffic to the CNS and enter a unique cytokine microenvironment that has the potential to influence the effector and/or regulatory mechanisms of these infiltrating cells. This project was initiated to determine how cytokines in the CNS microenvironment influence the pathogenicity of myelin-specific Th1 effector cells. Transforming growth factor (TGF)-β is a cytokine expressed in both the healthy and inflamed CNS with demonstrated suppressive effects on naïve CD4+ T cell differentiation; however, the effects of this cytokine on Ag-experienced Th1 effector cells are not well defined. Using myelin-specific T cell receptor (TCR) transgenic (Tg) mice in the experimental autoimmune encephalomyelitis (EAE) model of MS, we demonstrate that TGF-β elicits differential effects on proliferation, cellular activation, and cytokine secretion based on the differentiation state of a CD4+ T cell (i.e. naïve versus effector-memory). Additionally, Th1 effector cells begin to produce IL-10 as a self-regulatory mechanism that is enhanced with both activation in an environment rich in TGF-β, and repetitive Ag stimulation. TGF-β signaling caused direct binding of smad4 to the IL-10 promoter, providing molecular evidence for TGF-β-mediated regulation of IL-10 production in Th1 effector cells. Furthermore, the IL-10 produced by IFN-γ+IL-10+ Th1 effector cells decreased EAE severity by reducing the expression of encephalitogenic markers on IL-10R+ cells and preventing host-cell recruitment to the inflamed CNS. These data establish a regulatory mechanism by which highly activated Th1 effector cells modulate their pathogenicity through induction of IL-10. This work is a demonstration of the complex signaling networks that direct the functions of immune cells. Furthermore, the data presented highlight the importance of regulatory mechanisms that modulate the immune response, whether during normal inflammation or autoimmunity. In the context of MS, increasing the inherent regulatory potential of pathogenic effector-memory T cells may lead to novel therapies for the treatment of this debilitating disease.

Bone Morphogenetic Proteins (BMPs) are pleiotropic cytokines that play integral roles in embryogenesis, osteogenesis, and organogenesis. BMPs belong to the transforming growth factor-β (TGF-β) superfamily and are reported to function as tumor suppressors in early preneoplastic lesions of the prostate and tumor promoters in late stage prostate adenocarcinoma. Moreover, BMPs have been recently shown to be pivotal in controlling prostate tumorigenesis, and loss of BMP receptor function has been correlated to a higher Gleason grade in prostate cancer patients. During advanced prostate cancer, reports have shown that the IGF-I/PI3K/Akt/mTOR pathway is hyperactive in 50% of patients examined. In this light, we provide evidence that support that the IGF-I signaling axis inhibits BMP4-induced apoptosis, Smad-mediated gene expression, and BMP specific downstream targets. Suppression of the BMP4 signaling by IGF-I was reversed by direct genetic manipulation using enforced expression of wt-PTEN or DN-PI3K, use of chemical inhibitors against PI3K/Akt/mTOR, or small hairpin RNA-mediated silencing of mTORC1/2 subunits Raptor or Rictor. Our results support that IGF-I blocks BMP-induced transcription of Id1, Id2, and Id3, which are all downstream targets of BMP, through a PI3K/Akt/mTOR-dependent mechanism. Using various rat and human prostate epithelial cell lines as well as human prostate pathological specimens, we provide the first evidence that mTOR mediates inhibition of BMP-induced Smad1/5/8 activation. Deregulation of mTOR-mediated inhibition of BMP signaling pathway may be crucial to halting progression of prostate cancer formation. Furthermore, we report that direct inhibition of mTOR by rapamycin or rapalogs enhances BMP-mediated Smad1 and Smad5 activation in human prostate cancer cell lines and tissue. Utilization of lentivirus-based silencing and retroviral-based overexpression enabled us to show that Smad1 and Smad5 mediate rapamycin-induced cell death and activation of Id1. On the other hand, we showed that Smad8 represses rapamycin’s action in human prostate cell lines. All in all, the studies described here provide novel implications in the BMP and mTOR signaling axis in prostate cancer and new potential targets for the therapeutic intervention of this malignancy.

The unique material properties of skeletal matrices are critical for musculoskeletal tissue function. For example, bone matrix material properties (BMMPs) - including elastic modulus, hardness, and fracture toughness - reflect the ability of bone matrix to resist deformation and catastrophic failure. However, the mechanisms by which these properties are established remain unclear. Many reports support a role for TGF-&beta and Smad3 in regulating the skeletal matrix. Thus, we hypothesized that Smad3 is essential for maintaining the extracellular matrix in bone, cartilage, and tendon.Previously, our lab determined that TGF-&beta regulates BMMPs in development by signaling through its intracellular effector, Smad3. Here, we showed that BMMPs could also be regulated by pharmacologically disrupting TGF-&beta signaling. To further dissect the mechanism of BMMP regulation, we identified the downstream targets of TGF-&beta signaling. In vitro studies showed that TGF-&beta inhibits osteoblast differentiation by Smad3 repression of Runx2 function. Using nanoindentation, we found that TGF-&beta regulation of BMMPs through Smad3 is also Runx2-dependent. We further show that osteopontin, a matrix protein regulated by both TGF-&beta and Runx2, is essential for normal bone matrix material properties. Our results establish a mechanism through which TGF-&beta regulates BMMPs to maintain healthy bone. This suggests a paradigm in which matrix material properties are established by growth factors through their regulation of lineage-specific transcription factors. Indeed, our further studies in cartilage and tendon indicate an essential role for TGF-&beta/Smad3 signaling in regulating cell lineage-specific transcription factor expression and activity as well as matrix structure and composition. We anticipate that our research will lead to the identification of novel diagnostic strategies and therapeutics for the prevention or treatment of skeletal diseases, as well as aid in the development of improved biomaterials for tissue regeneration.

Tissue microenvironment is an important determinant of carcinogenesis. We demonstrate that ionizing radiation, a known carcinogen, affects cancer frequency and characteristics by acting on the microenvironment. Using a mammary chimera model in which an irradiated host is transplanted with oncogenic Trp53 null epithelium, we show accelerated development of aggressive tumors whose molecular signatures were distinct from non-irradiated hosts. Molecular and genetic approaches show that TGF-beta mediated tumor acceleration; molecular signatures implicated TGF-beta and genetically reducing TGF-beta abrogated the effect on latency. Surprisingly, tumors from irradiated hosts were predominantly estrogen receptor negative. This effect was TGF-beta independent and linked to mammary stem cell activity. Thus the irradiated microenvironment affects latency and clinically relevant features of cancer through distinct and unexpected mechanisms. Compared to sporadic breast cancer, women treated with radiation for childhood cancers are diagnosed with early onset breast cancer that is more likely to be estrogen receptor negative and have a worse prognosis. Our mammary chimera model shows that host irradiation alone can reduce latency, increase aggressive tumor growth and promote estrogen receptor-negative cancers. Thus changes to the stromal microenvironment rather than DNA damage accounts for many of the features that are observed in radiation-preceded breast cancer. We combined molecular and genetic approaches to identify distinct mechanisms via TGF-beta activity and stem cell deregulation. Our study further shows that host biology significantly alters cancer molecular signatures and such microenvironmental changes are an important biological conduit for cancer risk in humans.

Molecules present in the bone marrow microenvironment contribute to attraction, adhesion, and infiltration of circulating breast cancer cells. Breast cancer stem cells, characterized as CD44+/CD24-/low, are implicated in progression of the disease. However, the specific mechanism used by breast cancer cells to metastasize is unknown. This study thus aimed to determine the role of breast cancer stem cells and microenvironmental factors in bone marrow metastasis, specifically in the adhesion of migrating cancer cells to bone marrow endothelium. The focal points of the study were E-selectin, SDF-1α, and their binding partners because of their integral roles in the cell adhesion and migration of other cell types to bone. Both stem cell-like and non stem cell-like breast cancer cells were found to express CXCR4 which binds SDF-1α. SDF-1α consistently enhanced E-selectin related adhesion of breast cancer cells to endothelium. Conversely, TGF-β1 induced epithelial mesenchymal transition to make cancer cells stem-like appeared to have no effect on this process. These results indicate that the breast cancer stem cell phenotype may not be necessary for E-selectin mediated cancer homing to the metastatic site, but may play a role in a unique homing method to bone marrow or in the formation of a new tumor upon bone marrow infiltration.

TGFΒ induces epithelial-mesenchymal transdifferentiation (EMT) accompanied by cellular differentiation and migration, a process fundamental during embryonic development and one that is reactivated in a variety of diseases including fibrosis and cancer. Despite extensive transcriptomic profiling, identification of TGFΒ-inducible, EMT-specific genes has met with limited success. Here, we report a novel post-transcriptional pathway by which TGFΒ modulates expression of EMT-specific proteins and EMT itself. We show that heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) binds a structural, 33 nucleotides (nt) TGF-beta-activated translation (BAT) element in the 3'-untranslated regions (UTRs) of disabled-2 (Dab2) and interleukin-like EMT inducer (ILEI) transcripts, and repress their translation. TGFΒ activation leads to phosphorylation at Ser43 of hnRNP E1 by protein kinase Bβ/Akt2, inducing its release from the BAT element and reversal of translational silencing of Dab2 and ILEI mRNAs. Further, using a genome-wide combinatorial approach involving polysome profiling and RIP-Chip analyses we have identified a cohort of four mRNAs (Rhox5, Ube3A, Prl2c4 and IL-11Rα2) that follow the same pattern of regulation as Dab2 and ILEI. Each of the identified targets mRNA harbors a functional BAT element in the 3'-UTR and is required for TGFΒ-induced EMT. Modulation of hnRNP E1 expression or its post-translational modification alters TGFΒ-mediated translational activation of the target transcripts and EMT in vitro and in vivo. This cohort of mRNAs may represent a new TGFΒ responsive and hnRNP E1-mediated posttranscriptional regulon that regulates TGFΒ-induced EMT during development and metastatic progression of tumors in a temporal and expedited fashion. The autocrine response of cells to TGFΒ-induced Akt2 activation and subsequent translational activation of EMT inducer transcripts may represent a novel mechanism through which the increased TGFΒ expression in tumor cells contributes to cancer progression.

The World Heath Organization estimates that 15 million people worldwide have a stroke annually with many of these individuals suffering from an ischemic attack that will lead to their permanent disability or death. One approach for developing targeted stroke therapies is to identify endogenous responses to cerebral ischemia and determine whether their addition promotes tissue survival. The studies in this thesis focus on the effects of activin A in cerebral ischemia. In this study, experimental cerebral ischemia and reperfusion resulted in the rise of activin βA mRNA in the ischemic hemisphere and increased phosphorylated smad2/3 signals in neurons adjacent to the site of injury. In a defined cell culture model that uses hydrogen peroxide (H2O2)-free radical stress, activin A addition protected cortical neurons against oxidative challenge. H2O2 treatment increased neuronal activin βA mRNA expression and inhibition with neutralizing antibodies to activin A resulted in robust neuronal death. These studies indicate that cells respond to cerebral ischemia with increased activin βA expression and activin A addition promotes the survival of cortical neurons. Studies investigating the effects of added activin A after cerebral ischemia and reperfusion in vivo revealed a robust tissue protective effect. Intraventricular administration of activin A prior to stroke onset protected against ischemia-induced tissue death and provides evidence that activin A promotes tissue survival in ischemic stroke. Analysis of activin A treatment 6 hours after ischemia and reperfusion revealed that despite delayed administration, activin A effectively reduced tissue death at 24 and 72 hours and improved neurobehavior. Moreover, activin A treatment spared neurons within the ischemic hemisphere and led to a concomitant reduction in microglial activation. Activin A resulted in reduced phosphorylation of the stress-responsive signals, p38 and c-jun N-terminal kinase (JNK), members of the mitogen activated protein kinase (MAPK) family, both of which are implicated in neuronal apoptosis. Taken together, these novel data demonstrate that activin A is an endogenous response to cerebral ischemia and reperfusion injury and its addition promotes tissue survival after ischemic stroke.

Cervical cancer is the 2nd most common cancer affecting women worldwide (Chen 1999). This is alarming because cervical cancer is completely treatable and many times preventable. Previous epidemiological study in 1999 found that six of their 16 cervical cancer patients had blood genotypes with a mutation on the gene coding for the type-one receptor for transforming growth factor β (Chen 1999). This three alanine deletion in a nine alanine repeat mapped to the 9q22 chromosome on the exon 1 coding region of TGFBR1 and has now been described as TGFBR1*6A or *6A (Pasche 1998). The more common allele is known as TGFBR1*9A or *9A. The cytokine involved in TGFBR1*6A is TGFβ and the transforming growth factor β family is commonly known as a tumor suppressor gene pathway Further study has found the *6A polymorphism was of higher incidence in cancer patients of many types: colorectal, breast, ovarian, and head & neck; and metanalyses have shown that TGFBR1*6A is a tumor susceptibility allele (Pasche 2004). It has been hypothesized that if presence of *6A predisposes the patient to cancer, it may occur more often in the tumors themselves (Pasche 2005). This is referred to as, somatic acquisition, the mutational act of a cancer patient who has the wild-type 9A/9A blood genotype, and their tumor mutates to acquire the *6A polymorphism. This process has previously been shown in head & neck and colorectal cancers (Pasche 2005). Cervical cancer is similar to these cancers because it also is of epithelial origin. Therefore, we investigated the incidence of somatically acquired TGFBR1*6A in cervical cancer tumors of blood genotyped 9a/9a homozygotes. 119 tumor samples were genotyped which had been patient matched from the Midwestern cervical cancer cohort who had previously been identified as having a blood genotype of 9A/9A homozygote by our laboratory. Seven of the 119 (5.88%) patients showed evidence of somatic acquisition after PCR amplification and genotyping. Furthermore, one of the 119 (0.84%) was found to have a tumor genotype of 6A/6A.

Abstract Obesity is a disorder affecting many Understanding disorders in metabolism requires the identification of molecular and neural circuits underlying energy balance. Here we investigate the coordination between feeding behavior and fat storage in response to changing environmental conditions in C. elegans. The TGF-β ligand DAF-7 is expressed in two sensory neurons and is responsive to environmental conditions such as food availability and population density. Decreases in TGF-β signaling lead to increased fat storage, decreased feeding rate, and in young animals' entry into a hibernating dauer state. Here we show that DAF-7 signals to two interneurons to regulate fat storage, feeding, and dauer formation. However, within these interneurons different molecular signals independently regulate feeding, fat storage, and dauer formation. TGF-β signaling inhibits fat storage through G-protein signaling, despite increasing feeding through the neurotransmitters tyramine and octopamine. We also identify ~20 additional C. elegans genes that regulate feeding behavior and fat storage. Taken together, this work elucidates the complex neural and molecular circuits coordinating metabolism and behavior in nutrient limiting conditions.

BK virus (BKV) is a member of the polyomavirus family that infects nearly the entire human population at an early age. Following a subclinical primary infection, BKV is able to establish a persistent infection in the kidney and urinary tract. Reactivation of BKV occurs in immunocompromised individuals and can lead to severe disease. BKV-associated diseases include polyomavirus nephropathy (PVN), a form of acute interstitial nephritis, and hemorrhagic cystitis (HC), an infection of the bladder characterized by inflammation and hematuria. PVN and HC are increasing in prevalence, likely due to the development of more potent immunosuppressive therapies. To better understand which components of the immune system are important for regulating BKV infection, we began to examine the effect of cytokines on virus replication. IFN-gamma has a strong inhibitory effect on BKV transcription, gene expression, and replication, but does not affect the kinetics of the viral life cycle or trafficking of the virus to the nucleus. IFN-gamma treatment inhibited gene expression of three different BKV strains similarly, suggesting that regulation by this cytokine is relevant for all viral strains. We have begun to further examine regulation of viral transcription by IFN-gamma to identify the specific factors involved and investigate changes in viral chromatin structure. In contrast to IFN-gamma, TGF-beta has an upregulatory effect on BKV strain TU early promoter activity. Three other strains examined were either unaffected or downregulated by TGF-beta treatment in a cell type-dependent manner. The TGF-beta response elements were mapped within the promoter of BKV TU. The viral strain- and cell type-dependent effects of TGF-beta demonstrate the complex nature in which BKV is regulated by cytokine signaling. Finally, we have begun to investigate the inability of archetype strains of BKV to replicate in tissue culture. Overall, our findings will help us to better understand BKV persistence in healthy individuals and reactivation in immunocompromised patients.

Lubrication of cartilage involves a variety of physical and chemical factors, including lubricin, a synovial glycoprotein shown to be a boundary lubricant. It is unclear how lubricin boundary lubricates a wide range of bearings from tissue to artificial surfaces, and if the mechanism is similar for soluble and bound lubricin. The objective of this research was to investigate lubricin as a mediator of articular cartilage boundary mode frictional properties. The central hypothesis of this dissertation was that the boundary mode friction coefficient is mediated by localization of lubricin at the tissue surface. In order to determine the frictional properties of cartilage, a linearly oscillating cartilage-on-glass friction apparatus was designed and validated, and a novel extension of the Stribeck curve, a "Stribeck surface," was created to map cartilage lubrication modes. The first aim identified the ability of exogenous lubricin to lubricate articular cartilage and characterized the differential roles of bound and soluble lubricin. The second aim investigated the ability of endogenously synthesized lubricin to lubricate cell-alginate constructs generated from chondrocytes, meniscal fibrochondrocytes, and mesenchymal stem cells. The last aim determined the functional effects of short term exposure to TGF-beta, IL-1beta, and OSM on the frictional properties of cartilage explants. These studies demonstrate that molecular modification of the tissue surface by localization or removal of lubricin altered the boundary mode frictional properties of cartilage. Additionally, localization of lubricin at the tissue surface is not a simple adsorption mechanism, with aggregation, steric arrangement of the molecule, and electrostatic interactions possibly playing a role in lubricin's boundary lubricating ability. These findings point to two distinct mechanisms by which lubricin lubricates, one mechanism involving lubricin bound to the tissue surface and the other involving lubricin in solution. The results presented herein suggest that production of extracellular matrix capable of localizing lubricin may be just as or more important than lubricin production in the lubrication of engineered tissues. Further, the recovery of lubrication for catabolically exposed tissue by exogenous lubricin may be therapeutically important to combat high tissue friction found in injury and disease.

CD4+CD25+ T regulatory cells represent an important subset of lymphocytes whose function is to suppress autoimmune disease and control normal immune responses. There is much research indicating a direct role for TGF-beta expressed on the surface of Tregs in the suppressor function of these cells. TGF-beta, whether bound to the cell surface or secreted, is produced as part of a complex that renders the cytokine inactive. Thrombospondin is the primary activator of biologically latent TGF-beta. This research demonstrates that thrombospondin is expressed on the surface of T lymphocytes isolated from the blood and lymph nodes of normal and FIV positive felines. Thrombospondin is expressed at significantly higher levels on CD4+CD25+ T lymphocytes, but can be induced in culture by activating T helper cells in the presence of LPS and IL2. We also observed that the CD4+CD25- T helper cells isolated from FIV negative control lymph nodes were able to markedly upregulate surface thrombospondin expression compared with similarly stimulated CD4+CD25- T helper cells from FIV positive sources. These findings are initial steps in working to understand the mechanism behind latent TGF-beta activation on CD4+CD25+ T regulatory cells and the role this cell type plays in FIV/HIV pathogenesis.

Over one billion people worldwide are infected with blood-feeding intestinal hookworms. The life cycle of A. duodenale (humans) and A. caninum (dogs) includes the propensity for L3 to undergo a temporary state of developmental arrest in the host. In female hosts, tissue-arrested L3 reactivate during pregnancy and are transmitted to neonates through milk. During pregnancy, transforming growth factor (TGF)-β2 is upregulated in the mammary gland. Studies in C. elegans show that TGF-β and insulin-like signaling pathways regulate larval arrest and reactivation. We previously utilized an in vitro assay to demonstrate that recombinant human TGF-β stimulates a feeding response in tissue-arrested A. caninum L3. We hypothesize that host expression of TGF-β and pregnancy hormones such as estrogen and prolactin signal arrested L3 to reactivate. To facilitate in vivo analyses, we utilized a mouse model of A. caninum infection. Mice were utilized because L3 do not develop into adults but arrest in somatic tissues, and reactivate during the periparturient period. We investigated TGF-β1, TGF-β2 and IGF-1 transcript and serum cytokine profiles during late pregnancy, early lactation and mid-lactation to correlate levels with transmammary transmission of L3 to nursing pups. An in vitro co-culture system was developed to mimic in vivo conditions and assess effects of TGF-β and, estrogen and prolactin on larval reactivation. A. caninum L3 were co-incubated with skeletal muscle or mammary epithelial cells and larval reactivation was measured. Additionally, immune responses were assessed as by measuring serum and transcript levels of IFN-γ and IL-4 in skeletal muscle, mammary gland and spleen to dissect the effects of pregnancy and A. caninum infection in the mouse. Our findings suggest that host-derived TGF- β1 and IGF-1, but not TGF- β2, are important in reactivation and transmission of arrested A. caninum larvae. Also, a Th2-like response characterized by increased IL-4 transcript levels was observed in skeletal muscle, while a mixed Th1⁄Th2 profile was observed in mammary gland when comparing infection with A. caninum versus pregnancy/lactation in BALB⁄c mice.

Cited2 [CBP/p300 interacting transactivator with E (Glutamic acid) and D (Aspartic acid) rich C-terminal domain, 2] is a CBP/p300 binding transcription factor without typical DNA binding domains. Cited2 interacts with LIM-homeobox gene 2 (Lhx2), Transcription factor AP2 (TFAP2), and nuclear receptors to function as a transcriptional co-activator. Cited2 is implicated in the control of cell growth and malignant transformation in Rat1 cells. Cited1 enhances Smad mediated transcription, suggesting that members of the Cited family may function as a co-activator in transforming growth factor-beta (TGF-beta) signaling. We have explored the function of Cited2 in the TGF-beta signaling pathway. In promoter reporter assays, Cited2 enhances Smad3 mediated transcription. This may occur through a direct physical association of Cited2 with Smads 2/3 and p300. We found that Cited2 modulates TGF-beta mediated upregulation of Matrix Metalloproteinase 9 (MMP9). In chromatin immunoprecipitation experiment, Cited2 and Smad3 are recruited to MMP9 promoter upon TGF-beta stimulation. Knockdown of Cited2 in MDA-MB-231 cells attenuates TGF-beta mediated cell migration, suggesting that Cited2 could play a role during tumor progression. Cited2 is downregulated by TGF-beta in MDA-MB-231 cells. We show that Smad2/3/4 and p38MAPK pathways are involved in TGF-beta mediated downregulation of Cited2. Nuclear run-on analysis and Cited2 promoter/reporter assays demonstrate that downregulation of Cited2 by TGF-beta is through posttranscriptional regulation. In transcriptional inhibitor assays, Cited2 mRNA turnover is increased under TGF-beta stimulation. We examined the expression of recombinant Cited2 gene introduced into MDA-MB-231 cells by stable transfection, and found that mRNA containing the Cited2 protein-coding region controlled by a heterologous promoter indeed responds to TGF-beta mediated downregulation. Our findings support that TGF-beta mediated downregulation of Cited2 is under posttranscriptional control, through Smad and p38MAPK pathways, and requires the presence of its coding sequence. The results of this thesis research provide supporting evidence of Cited2 as a transcription modulator in TGF-beta signaling and also suggest a feedback mechanism that downregulation of Cited2 by TGF-beta attenuates the expression of TGF-beta responsive genes such as MMP9 and MMP13. As MMP9 and MMP13 are highly involved in tumorigenesis, our research proposes that Cited2 could be a potential anticancer target.

The transforming growth factor-beta (TGFbeta) family of signaling ligands encompasses a group of highly conserved proteins that play critical roles in metazoan development and physiology. TGFbeta family ligands signal through a membrane complex composed of TypeI and TypeII receptors, which transduce intracellular signals via Smad proteins. Activated Smad proteins act as promoter-specific transcription factors to induce changes in cellular growth and differentiation. Inhibin is an atypical member of the TGFbeta family, which selectively inhibits signaling by other TGFbeta family members. Inhibin is classically understood to antagonize signaling by activin, a canonical TGFbeta ligand that signals through Smad2 and Smad3. Opposing interactions between inhibin and activin regulate the function of the hypothalamic-pituitary-gonadal (HPG) axis and steroidogenic cell proliferation in the gonads. Inhibin-null (Inha-/-) mice display defects in both processes, exhibiting elevated follicle stimulating hormone (FSH) levels and estrogen-secreting gonadal tumors. Inha-/- mice develop estrogen-secreting adrenocortical neoplasms upon surgical gonadectomy, indicating that inhibin also regulates adrenocortical cell growth. The work presented in this thesis has attempted to elucidate several aspects of gonadal and adrenal tumorigenesis in Inha-/- mice, including the cellular identity of these neoplasms and signaling mechanisms involved in tumorigenesis. My studies have revealed that the adrenocortical neoplasms in Inha-/- mice are derived from misspecified adrenocortical stem/progenitor cells, which undergo a change in cellular fate to form gonadal tumors in the adrenal. These tumors have similar steroidogenic and transcriptional profiles to Inha-/- gonadal tumors, suggesting that the tumor-suppressing mechanism of inhibin is conserved between the gonads and adrenal. This assertion is supported by the common dependence of both tumor types on Smad3, which is required for efficient tumor cell proliferation. Genetic removal of Smad3 from Inha-/- mice disrupts mitogenic signaling by both activin and follicle stimulating hormone, and dramatically attenuates disease progression in the adrenal and gonad. Despite this similarity, tumorigenesis in the Inha-/- adrenal cortex may also involve unique mechanisms independent of activin signaling. My studies particularly implicate a novel interaction between inhibin and TGFbeta2 in adrenocortical tumor initiation, suggesting that inhibin may play multiple complex roles in cell fate determination and cellular growth suppression in steroidogenic tissues.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-ƒ× may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.