<p>The specification of a single axon and multiple dendrites is the first observable event during neuronal morphogenesis and such structural specialization underlies neural connectivity and nervous system function. Numerous intracellular signaling components that are required for axon specification have been described but how such signaling paradigms are initiated by extracellular factor(s) within the embryonic milieu is poorly understood. Here, I describe how transforming growth factor-&beta; (TGF-&beta;), an embryonic morphogen that directs structural plasticity and growth in various cell types, initiates signaling pathways both in vivo and in vitro to fate naïve neurites into axons. Using conditional knockout strategies, I found that cortical neurons lacking the type II TGF-&beta; receptor (T&beta;R2) fail to initiate axons during development, and interestingly, fail to engage radial migration. In cultured neurons, exogenous TGF-&beta; is sufficient to direct the rapid growth and differentiation of an axon and genetic enhancement of receptor activity promotes the formation of multiple axons. The cellular polarization of receptor activity occurs through the interaction of the type-I TGF-&beta; receptor with Par6, a component of the axon-specifying Par3/Par6 polarity complex. Receptor distribution is restricted to axons, and downstream signaling events required for axon specification are triggered when Par6 is phosphorylated by T&beta;R2. Together, these results indicate that TGF-&beta; is the extrinsic cue for neuronal polarity in vivo and directs neuronal polarity by controlling Par6 activity and cellular migration during axon generation.</p> / Dissertation

<p>Breast cancer remains among the most common cancers of the developed world. Despite advances in treatment modalities, deaths due to breast cancer are the second leading cause of cancer death among women. The transforming growth factor-beta (TGF-&beta;) pathway is an important modulator of breast cancer progression, acting in a tumor suppressing fashion in early carcinogenesis but switching in a poorly understood fashion to a promoter of cancer progression in later stages. Mutations and loss of function of TGF-&beta; components are common across a variety of cancers. In particular, the expression of the type III TGF-&beta; receptor (T&beta;RIII) is decreased with cancer grade and clinical progression in prostate, lung, ovarian, and pancreatic cancers. In an effort to enhance our understanding of the biology of TGF-&beta; on carcinogenesis, this dissertation looks at the role of T&beta;RIII in breast cancer progression.</p><p>Through an examination of clinical specimens, loss of T&beta;RIII was seen at both the message and protein levels with increasing tumor grade. Analysis of correlated patient outcomes showed that low T&beta;RIII expression was predictive of a shorter time to recurrence, demonstrating clinical relevance for T&beta;RIII expression. The contribution of T&beta;RIII to tumor progression was further examined by examining known TGF-&beta; functions, including proliferation, apoptosis, migration, and invasion. T&beta;RIII had no effect on proliferation or apoptosis, but had a suppressive effect on metastasis <italic>in vivo</italic>, as mammary cancer cells stably expressing T&beta;RIII that were orthotopically injected exhibited lower metatstatic burden and local invasion. <italic>In vitro</italic>, breast cancer cells exhibited suppression of migration and invasion in transwell assays. Finally, soluble T&beta;RIII (sT&beta;RIII) was shown to recapitulate the suppressive effects on invasion.</p><p>To further explore other potential mechanisms by which T&beta;RIII may be mediating its tumor suppressive effects, I examined the contribution of the cytoplasmic domain of T&beta;RIII, which is known to be critical in the regulation of T&beta;RIII cell surface expression and downstream signaling. <italic>In vitro</italic>, I demonstrated that abrogation of the cytoplasmic domain attenuates the T&beta;RIII-mediated suppression of migration and invasion. T&beta;RIII's suppressive effects are also concomitant with loss of TGF-&beta; signaling, as abrogation of the cytoplasmic domain failed to attenuate TGF-&beta; signaling while the full length receptor was able to do so. <italic>In vivo</italic>, I also showed that in the absence of the cytoplasmic domain, T&beta;RIII is unable to suppress metastasis and local invasion. Finally, a closer dissection of the cytoplasmic domain revealed that abolishing the interaction of T&beta;RIII with the scaffolding protein GIPC also attenuated T&beta;RIII's ability to dampen TGF-&beta; signaling and invasion.</p><p>In sum, T&beta;RIII was established as a prognostic marker for recurrence-free survival of breast cancer patients and as a suppressor of metastasis, migration, and invasion. Furthermore, several mechanisms contribute to T&beta;RIII's tumor suppressive effects, namely the generation of sT&beta;RIII and the interaction of T&beta;RIII with GIPC. Taken together, these studies further demonstrate the importance of TGF-&beta; signaling in cancer biology, elucidate mechanisms by which T&beta;RIII suppresses breast carcinogenesis, and expand upon our understanding of the emerging roles of T&beta;RIII in regulating tumor biology in general.</p> / Dissertation

Articular cartilage functions to maintain joint mobility. The loss of healthy, functional articular cartilage due to osteoarthritis or injury can severely compromise quality of life. To address this issue, cartilage tissue engineering approaches are currently in development. Bone marrow-derived mesenchymal progenitor cells (MPCs) hold much promise as an alternative cell source for cartilage tissue engineering. While previous studies have established that MPCs from humans and multiple other species undergo in vitro chondrogenic differentiation, additional research is needed to define conditions that will enhance MPC differentiation, increase matrix production by differentiating cultures, and support development of functional tissue-engineered cartilage constructs. Mechanical loading may be an important factor regulating chondrogenic differentiation of MPCs and cartilage matrix formation by chondrogenic MPCs. This thesis work evaluated the influence of oscillatory unconfined compressive mechanical loading on in vitro MPC chondrogenic activity and biosynthesis within hydrogel suspension. Loading was conducted using MPCs cultured in media supplements supporting chondrogenic differentiation. Possible interactions between the number of days in chondrogenic media preceding loading initiation and the ability of the MPC culture to respond to mechanical stimulation were explored in two different loading studies. The first loading study investigated the effects of 3 hour periods of daily oscillatory mechanical stimulation on subsequent chondrogenic activity, where chondrogenic activity represented an assessment of cartilage matrix production by differentiating MPCs. This study found that oscillatory compression of MPCs initiated during the first seven days of culture did not enhance chondrogenic activity above the level supported by media supplements alone. The second loading study evaluated changes in biosynthesis during a single 20 hour period of oscillatory mechanical stimulation to assess mechanoresponsiveness of the MPC cultures. This study found that MPCs modulated proteoglycan and protein synthesis in a culture time-dependent and frequency-dependent manner upon application of oscillatory compression. Together the two loading studies provide an assessment of dynamic compressive mechanical loading influences on MPC cultures undergoing chondrogenic differentiation. The information gained through in vitro studies of differentiating MPC cultures will increase basic knowledge about progenitor cells and may also prove valuable in guiding the future development of cartilage tissue engineering approaches.

The incidence rate of chronic hepatopathy in Taiwan is high, which afflicts the patients by progressively developing irreversible cirrhosis. Hepatic fibrosis is the intermediate and crucial stage of this process, characterized by reversibility. If treated properly in this stage, cirrhosis can be successfully prevented. In the liver, activated stellate cells are the key mediators of fibrosis. Transforming growth factor-

Recent statistics reveal breast cancer as the most common cancer among women and accounts for approximately 41,000 mortalities per year. In diagnosis, features such as stage, grade, lymph node metastasis are important prognostic indicators that help guide physicians and oncologist towards optimal patient care. Presence of established pathological markers such as ER, PR, and Her2/neu status would indicate ideal adjuvant therapy situation. Although treatment of these types of breast cancer is well established, cancer that lack all three receptors, “triple negatives” or “basal like” do not respond to adjuvant therapy and are considered more aggressive in that patients tend to recur early and experience visceral metastasis. Although scientists have uncovered numerous molecular biology mechanisms in search of an understanding in cancer, leading to development of fields such as apoptosis or growth pathways; cell cycle; angiogenesis; metastasis; and more recently cancer stem cells, much work remains as cancer is still not eradicated. MicroRNAs (miRNAs) are post transcriptional regulators of gene expression. Their discovery and functional understanding have only been uncovered in the past ten years. Long pri-miRNAs are transcribed from the genome and processed into premiRNAs by Dicer; and then into short single stranded mature miRNAs complexed with Argonaute proteins to inhibit protein translation. The first link of miRNAs to cancer was made only relatively recently, but the field has expanded exponentially since. TGF- β induced Epithelial to Mesenchymal Transition model in Normal Mouse Mammary Gland Epithelia Cells (NMuMG) is a commonly used model to dissect the molecular processes of breast cancer metastasis. Using miRNA microarray, we demonstrated miR-155 was upregulated along with alterations of other miRNAs. This observation was validated with Northern and qRT-PCR analysis. Promoter and ChIP analysis revealed TGF- β activated the Smad4 transcriptional complex to induce the expression of miR-155. The reduction of RhoA protein levels by ubiquitination has been described to be a critical step during EMT, and we showed miR-155 down regulates RhoA proteins without degrading its mRNA levels; therefore, preventing de novo synthesis of RhoA proteins in the course of EMT. The interaction between miR-155 and RhoA’s 3’UTR was confirmed by reporter assays. In summary, we reported the importance of miR-155 during TGF β induced EMT in NMuMG cells. FOXO3a is a well studied tumor suppressor transcriptional factor and resides in the nucleus to transcribe pro-apoptotic genes such as Bim, or p27 in the active state. During conditions when cells are signaled to grow and divide, it is phosphorylated by oncogenes such as AKT or IKK β, becomes inactivated and translocates into the cytoplasm. We have shown for the first time that FOXO3a activity is also regulated by miRNAs, specifically miR-155. Western and Northern analysis revealed a correlation between FOXO3a protein and mature miR-155 RNA levels in breast cancer cell lines along with breast tumor and normal tissues. Specifically, miR-155 expression is low in BT474 and high in HS578T, and inversely correlates with endogenous FOXO3a protein levels. Overexpression of miR-155 decreased endogenous FOXO3a protein and knockdown of miR-155 HS578T rescued its expression. Reporter assay experiments validated the interaction between miR-155 and FOXO3a 3’UTR. More importantly, overexpression of miR-155 in BT474 protected the cells from apoptosis induced by drugs while knockdown of miR-155 in HS578T initiated cell death even in the absence of drugs. In summary, we have shown the importance of miR-155 in chemosensitivity by targeting FOXO3a in breast cancer. MiR-155 has been previously shown up-regulated in multiple types of malignancies, including breast cancer. In addition, miR-155 expression was reported to correlate very strongly to survival in lung and pancreatic cancer. We validated by qRTPCR and Northern analysis that miR-155 expression is detected only in breast tumors and not normal breast tissue. In situ hybridization of breast cancer tissue microarrays revealed similar results. In light of previous studies that showed a correlation between miR-155 and survival in lung and pancreatic cancers, we performed an X-tile analysis to determine an optimal cut point for miR-155 level in our breast cancer sample population that would correlate to ten years overall survival. Verification using Kaplan-Meier validated a cut point at 90.14 to significantly correlate to overall survival (P=0.007). In addition, Chi-square analysis revealed miR-155 expression to correlate with high tumor stage, grade and lymph node metastasis. However, miR-155 expression did not correspond to ER, PR, or HER2/neu status, but this is hardly surprising since computational analysis does not predict miR-155 to target these genes. In summary, we have shown deviant expression of miR-155 in breast cancer. Due to its correlation with overall survival; higher grade and stage; lymph node metastasis, and triple negative subtype, miR-155 may prove to be a valuable prognostic marker and therapeutic target for breast cancer intervention.

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.

Objective: Endothelial cell (EC)-induced mesenchymal cell (MC) differentiation toward a mural cell phenotype requires transforming growth factor beta (TGF-β), cell contact and connexin 43 (Cx43)- or Cx45- heterocellular gap junction intercellular communication (GJIC). However, the identity of the communicated signal, the features of Cx43 required, and the possible regulatory mechanisms have not been elucidated and were investigated herein. Methods & Results: To determine whether channel functionality and the major regulatory domain (the carboxyl terminus, CT) of connexin Cx43 are necessary to support EC-induced differentiation, Cx43 deficient MCs (incapable of undergoing EC-induced mural cell differentiation without re-expression of Cx43 or Cx45) were transduced with wild-type (Cx43wt), channel dead, or truncated (Cx43tr-residues 258-382 deleted) versions of Cx43 and their ability to support EC-induced differentiation was assessed. Our data indicate that both channel functionality and presence of the CT domain are both necessary for EC-induced mural cell differentiation. Moreover, expression of Cx40 did not restore ability of MCs to undergo EC-induced mural cell differentiation, despite supporting GJIC. To determine whether (and which) specific regulatory sites in the carboxyl terminus are necessary for EC-induced mural cell differentiation, constructs of Cx43 with serine to alanine substitutions at the mitogen activated protein kinase (MAPK) or protein kinase C (PKC) target sites were introduced into Cx43 deficient MCs and their ability to undergo EC-induced differentiation was tested. The data indicated that the MAPK targeted serines (S255,279,2982) are necessary, while the PKC targeted serine (S368) is dispensable, for this process. To determine whether calcium ions might be the messengers communicated between ECs and MCs, we investigated whether elevation in EC free intracellular calcium concentration (with ionomycin treatment) can replace Cx43-mediated GJIC, activate TGF-β and induce differentiation. Conclusions: Channel functionality, CT domain and the MAPK target sites in Cx43 are all necessary, and neither alone is sufficient, for Cx43-mediated, EC-induced mural cell differentiation. Unlike Cx43, Cx40 is not capable of supporting EC-induced differentiation, despite supporting GJIC. Calcium is unlikely to be the messenger critical to TGF-β activation during EC-induced differentiation, but similar signaling pathways can be initiated. Taken together, these data support a role for connexins in EC-induced differentiation that is complex and goes beyond that of a simple conduit.

<p>The bone morphogenetic protein (BMP) signaling pathways have important roles in embryonic development and homeostasis. BMPs have been shown to pattern the dorsoventral axis in zebrafish (<italic>Danio rerio</italic>) early during embryonic development by establishing a dorsal-to-­ventral ligand gradient. During tumorigenesis, BMPs primarily function as tumor promoters, as an increase in BMP expression is associated with an increase in invasion, migration, epithelial-­to-­mesenchymal transition (EMT), proliferation and angiogenesis.</p><p>Although it is clear that BMPs play multiple roles in these biological events, the precise mechanism by which BMPs mediate these functions is not fully understood. Canonically, BMP ligands signal through cell surface receptor complexes that phosphorylate transcription factors, Smad1, Smad5 and Smad8, which mediate BMP-­ specific gene transcription. While studying BMP signaling during cancer progression, we determined that BMPs unexpectedly signal through the canonical TGF-&beta;-­responsive transcription factors, Smad2 and Smad3.</p><p>We determined that BMP-­induced Smad2/3 signaling occurs preferentially in embryonic cells and transformed cells. BMPs signal to Smad2/3 by stimulating complex formation between the BMP binding TGF-­&beta; superfamily receptors, ALK3/6, and the Smad2/3 phosphorylating receptors, ALK5/7. BMP signaling through Smad1/5/8 induces MET, while Smad1/5 and Smad2 mediate dorsoventral axis patterning in zebrafish embryos and Smad3 facilitates invasion.</p><p>Taken together, our data provides evidence that BMP-­induced Smad2 and Smad3 phosphorylation occurs through a non-­canonical signaling mechanism to mediate multiple biological events. Thus, the signaling mechanisms utilized by BMPs and TGF-­&beta; superfamily receptors are broader than previously appreciated.</p> / Dissertation

The Snail1 transcriptional repressor is a key factor responsible in triggering epithelial to mesenchymal transition. Although Snail1 is widely expressed in early development, it is limited in adult animals to a subset of mesenchymal cells where it has a largely unknown function. In this project we have demonstrated that Snail1 is required to maintain mesenchymal stem cells (MSCs). This effect is associated to the responsiveness to TGF-[beta]1 which showed a strong Snail1 dependence. Snail1-depletion in conditional knock-out adult animals caused a significant decrease in the number of bone marrow-derived MSCs. In culture, Snail1-deficient MSCs prematurely differentiated to osteoblasts or adipocytes and, in contrast to controls, were resistant to the TGF-[beta]1-induced differentiation block. TGF-[beta]1 was unable to up-regulate most of its targets in Snail1 KO MSCs, an effect that was related, but not limited, to defective PTEN repression and Akt activation. Correspondingly, an analysis of human sarcomas also showed enhanced expression of Snail1 in undifferentiated tumors, which was strongly associated with high expression of TGF-[beta] and poor outcome. These results not only demonstrate a new role for Snail1 in TGF-[beta] response and MSC maintenance but also suggest the involvement of MSCs in sarcoma generation. / El repressor transcripcional Snail1 ha estat descrit principalment com el responsable de la inducció de la transició epiteli mesènquima. Encara que Snail1 s’expressa durant les etapes més primerenques del desenvolupament embrionari, la seva expressió en adults es veu limitada en un conjunt de cèl•lules mesenquimals sense saber-se la seva funció. En aquest projecte hem demostrat que Snail1 es requereix per mantenir el fenotip més indiferenciat de les cèl•lules mare del mesènquima. Aquesta funció la fa en part, per la capacitat de resposta de la citoquina TGF-[beta] la qual mostra una força dependència amb Snail1. Quan s’elimina Snail1 en ratolins adults provoca una clara disminució en el nombre de cèl•lules mare de la medul•la òssia. Aquestes cèl•lules en cultiu presenten una clara diferenciació prematura a osteoblasts i adipòcits. Pel contrari, tractaments amb TGF-[beta]1 aturen la diferenciació. El TGF-[beta]1 es incapaç de incrementar moltes dianes en cèl•lules mare del mesènquima aïllades del ratolí deficient per snail1, aquest efecte en part es degut a la repressió de PTEN i l’activació de AKT. L’anàlisi de sarcomes humans ens ha mostrat una alta expressió de Snail1, el qual també es troba associada amb una alta expressió de TGF-[beta] i baixa supervivència. Aquests resultats no només demostren una nova funció per Snail1 en resposta a TGF-[beta] i el manteniment de les MSC, sinó que també suggereix que Snail1 podria participar en la generació del sarcoma.

Chronic inflammatory conditions such as Crohn's disease (CD) and Ulcerative colitis (UC) are risk factors for colon cancer. TGFβ has been shown to be dysregulated in colon cancer. Bacteria-induced inflammation is necessary for the induction of colon cancer in TGFβ mouse models. However, the mechanism by which TGFβ regulates the inflammatory response in these models is not well elucidated. It was our thought that we needed to be able to distinguish what was TGFβ dependent and what was inflammation dependent. To do this we created 2 colonies of Smad3 mice. One colony was housed with normal colonic bacteria (Smad3-uninfected animals) and the other colony (Smad3-infected animals) had chronic H. hepaticus infection. As previously seen the Smad3⁻/⁻- infected animals developed colitis and carcinoma (~40%). In the absence of H. hepaticus infection SMAD3 was found to negatively regulate TLR4 expression. This was then exacerbated with the addition of H. hepaticus resulting extreme up-regulation of TLR4 and the downstream effectors IRAK4 and NF-κB in Smad3⁻/⁻-infected colonic tissues. Examination of adaptive immune regulation in this model demonstrated that SMAD3 was necessary for FOXP3 expression in H. hepaticus-infected splenocytes. Loss of SMAD3 resulted in up-regulation of IL17 and reduced iTreg populations. These data demonstrate the important role SMAD3 has in maintaining tolerance to microbial populations through both the innate and adaptive immune systems.