Der TGFβ-Signalweg ist ein multifunktionales System, das zelluläre Prozesse reguliert, die von Proliferation und Migration bis zu Differenzierung und Zelltod reichen. Nach Ligandenbindung und Rezeptoraktivierung translozieren SMAD-Proteine zum Zellkern und induzieren die Expression zahlreicher Zielgene. Während viele Komponenten des TGFβ-Signalweges identifiziert wurden, verstehen wir noch nicht genau, wie die Aktivierung des Signalwegs in verschiedene zelluläre Antworten übersetzt wird. Da die zelluläre Antwort auf einen gegebenen Stimulus oft sogar in genetisch identischen Zellen variiert, konzentrierte ich mich auf die Messung der Signalwegaktivität auf der Einzelzellebene. Durch die Kombination fluoreszierender Reporterzelllinien mit Zeitraffer-Lebendzellmikroskopie und automatisierter Bildanalyse beobachtete ich die zytoplasmatische und nukleäre Translokation von SMADs mit hoher zeitlicher und räumlicher Auflösung in Hunderten einzelner Zellen. Unsere Experimente zeigten, dass die Signalwegaktivität in eine erste synchrone Phase der SMAD-Translokation, gefolgt von einer Adaption und einer zweiten Signalphase mit hoher Variabilität in Stärke und Dauer der nuklearen Akkumulation unterteilt werden kann. Darüber hinaus beobachtete ich, dass Zellen, die aufgrund ihrer dynamischen Eigenschaften in Subpopulationen gruppiert sind, unterschiedliche phänotypische Reaktionen zeigen. Ich war nun daran interessiert, die Netzwerkinteraktionen zu identifizieren, die diese Dynamiken formen und fokussierte mich auf den Crosstalk mit nicht-kanonischen Komponenten des TGFβ-Signalweges. Ich konnte zeigen, dass die Hemmung der MAP Kinasen p38 und ERK die zweite Signalphase spezifisch aufhebt. Diese dynamische Remodellierung führt zu Veränderungen in der Zielgenexpression und den Zellschicksalen. Dies wird zu einem tieferen Verständnis der molekularen Netzwerke führen, die die TGFβ-Signaltransduktion regulieren und Möglichkeiten eröffnen, es in erkrankten Zellen zu modulieren. / The TGFβ pathway is a multi-functional signaling system regulating cellular processes ranging from proliferation and migration to differentiation and cell death. Upon ligand binding and receptor activation, SMAD proteins translocate to the nucleus and induce expression of numerous target genes. While many components of the TGFβ pathway have been identified, we are still challenged to understand how pathway activation is translated into distinct cellular responses. As the cellular response to a given stimulus often varies even in genetically identical cells, I focused on measuring pathway activity on the single cell level. By combining fluorescent reporter cell lines with time-lapse live-cell microscopy and automated image analysis, I monitored the cytoplasmic to nuclear translocation of SMADs with high temporal and spatial resolution in hundreds of individual cells. Our experiments demonstrated that pathway activity can be divided into a first synchronous phase of SMAD translocation, followed by adaptation and a second signaling phase with high variability in the extent and duration of nuclear accumulation. Furthermore, I observed that cells clustered into subpopulations according to their dynamic features show different phenotypic responses. I was interested in identifying the network interactions that shape these dynamics and focus on crosstalk with non-canonical components of the TGFβ pathway. I could show that inhibition of the MAP kinases p38 and ERK specifically abrogates the second signaling phase. This dynamic remodeling led to changes in target gene expression and cell fate decisions. I explored the molecular mechanisms underlying this interaction of the canonical and non-canonical pathways. This will provide a deeper understanding of the molecular networks regulating TGFβ signaling and open opportunities to modulate it in diseased cells.

Ovarian Cancer is the 5th most common cause of cancer death in women and the second most common gynaecological cancer in the UK. Worldwide, around 152,000 women were estimated to have died from ovarian cancer in 2012. Survival rates for women with epithelial ovarian cancer have not significantly changed since platinum-based treatment was introduced over 30 years ago. This is particularly disconcerting considering the fact that there is a less than 5% five year survival rate for patients diagnosed with late stage high grade serous ovarian cancer. This thesis examines the role of BMP signalling in ovarian cancer using in vitro cancer cell models. It builds upon the initial published work by the Inman lab identifying autocrine BMP9 as a promoter of ovarian cancer cell proliferation. The findings of Chapters 3-5 provide strong evidence indicating BMP9 as a context specific modulator of ovarian cancer cell proliferation. This significantly builds upon on the sole pro-proliferative BMP9 growth response previously described. Responding cell lines were subjected to a microarray with and without BMP9 treatment In order to determine early BMP target genes which were subsequently transiently knocked down in order to determine their role in the aetiology of said growth phenotype. ID1 gene expression was found to significantly contribute to the BMP9 proproliferative phenotype. Moreover several other BMP genes identified significantly alter basal cell proliferation. It was subsequently determined that BMP9 implemented a cell growth phenotype by negating apoptosis. .Excitingly, preliminary evidence suggests a marked reduction in detectable levels of a recently described Bax isoform, Bax β that coincide with BMP9 addition and the resultant anti-apoptotic phenotype observed. This is very interesting as no prior evidence correlating the BMP family and Bax β currently exists. These findings provide an enhanced understanding of BMP9s contribution to ovarian cancer pathogenesis that may result in the development of effective and targeted therapeutic interventions upon further stratification of the contextuality of the BMP induced growth response.

<p>TGF-β is the founding member of a multifunctional family of cytokines that regulate many aspects of cell physiology, including cell growth, differentiation, motility and death and play important roles in many developmental and pathological processes. TGF-β signals by binding to a heterotetrameric complex of type I and type II serine/threonine kinase receptors. The type I receptor is phosphorylated and activated by the type II receptor and propagates the signal to the nucleus by phosphorylating and activating receptor-regulated Smad proteins (R-Smads). Once activated, the R-Smads translocate to the nucleus together with the common partner Smad, Smad4, in heteromeric complexes and regulate transcription of target genes.</p><p>The cell cycle inhibitor p21^Waf1/Cip1 (p21) is induced by a number of factors including p53 and TGF-β, and its high expression is associated with cellular differentiation and senescence. Low levels of p21 are required for the propagation of the cell cycle, where high levels of p21 expression result to cell cycle arrest. The mode of action of p21 is by interacting with and dissociating cyclin E- and cyclin A-CDK complexes. p21 is very potently upregulated by TGF-β in cell types of epithelial origin and this sustained upregulation is of utmost importance for TGF-β to exert its growth inhibitory effect.</p><p>The aim of this study was to clarify the mechanisms by which the cell cycle inhibitor p21 is regulated during the TGF-β-induced cell growth inhibition. During the course of this work we established that TGF-β regulates p21 via the Smad pathway at the transcriptional level and that upregulation of the p21 levels cannot be achieved in the absence of proper Smad signaling. This regulation is achieved by Smad proteins interacting with the transcription factor Sp1 at the proximal <i>p21</i> promoter region. We also established that p21 is regulated by all the TGF-β superfamily pathways as we showed that all type I receptors of the superfamily are able to upregulate p21. Despite that, we demonstrated that p21 induction by other members of the superfamily, such as BMPs, is not sufficient for growth suppression. This is because BMPs regulate additional genes such as <i>Id2</i> that counteract the effect of p21 on cell growth. Furthermore, we examined the homeobox gene <i>Meox2</i>, which is regulated by TGF-β, and established that this factor is important for the sustained p21 regulation and the cell growth inhibitory program exerted by TGF-β. Simultaneously, we examined the cross-talk between Notch and TGF-β signaling pathways and established a synergy between Notch and TGF-β during epithelial cell growth inhibition. We showed that TGF-β-induced growth arrest requires intact Notch signaling. Abrogation of Notch signaling results in a blockage of sustained p21upregulation, required for the TGF-β-induced growth arrest to occur.</p><p>This work contributes substantially to the mechanism of both immediate-early and prolonged-late regulation of p21 by TGF-β-superfamily pathways, leading to cell growth inhibition of epithelial cells.</p>

TGF-β is the founding member of a multifunctional family of cytokines that regulate many aspects of cell physiology, including cell growth, differentiation, motility and death and play important roles in many developmental and pathological processes. TGF-β signals by binding to a heterotetrameric complex of type I and type II serine/threonine kinase receptors. The type I receptor is phosphorylated and activated by the type II receptor and propagates the signal to the nucleus by phosphorylating and activating receptor-regulated Smad proteins (R-Smads). Once activated, the R-Smads translocate to the nucleus together with the common partner Smad, Smad4, in heteromeric complexes and regulate transcription of target genes. The cell cycle inhibitor p21Waf1/Cip1 (p21) is induced by a number of factors including p53 and TGF-β, and its high expression is associated with cellular differentiation and senescence. Low levels of p21 are required for the propagation of the cell cycle, where high levels of p21 expression result to cell cycle arrest. The mode of action of p21 is by interacting with and dissociating cyclin E- and cyclin A-CDK complexes. p21 is very potently upregulated by TGF-β in cell types of epithelial origin and this sustained upregulation is of utmost importance for TGF-β to exert its growth inhibitory effect. The aim of this study was to clarify the mechanisms by which the cell cycle inhibitor p21 is regulated during the TGF-β-induced cell growth inhibition. During the course of this work we established that TGF-β regulates p21 via the Smad pathway at the transcriptional level and that upregulation of the p21 levels cannot be achieved in the absence of proper Smad signaling. This regulation is achieved by Smad proteins interacting with the transcription factor Sp1 at the proximal p21 promoter region. We also established that p21 is regulated by all the TGF-β superfamily pathways as we showed that all type I receptors of the superfamily are able to upregulate p21. Despite that, we demonstrated that p21 induction by other members of the superfamily, such as BMPs, is not sufficient for growth suppression. This is because BMPs regulate additional genes such as Id2 that counteract the effect of p21 on cell growth. Furthermore, we examined the homeobox gene Meox2, which is regulated by TGF-β, and established that this factor is important for the sustained p21 regulation and the cell growth inhibitory program exerted by TGF-β. Simultaneously, we examined the cross-talk between Notch and TGF-β signaling pathways and established a synergy between Notch and TGF-β during epithelial cell growth inhibition. We showed that TGF-β-induced growth arrest requires intact Notch signaling. Abrogation of Notch signaling results in a blockage of sustained p21upregulation, required for the TGF-β-induced growth arrest to occur. This work contributes substantially to the mechanism of both immediate-early and prolonged-late regulation of p21 by TGF-β-superfamily pathways, leading to cell growth inhibition of epithelial cells.

Activin-A is a member of transforming growth factor-β (TGF-β) superfamily of cytokines which includes TGF-βs, Activins, Nodal, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and anti-Mullerian hormone (AMH). TGF-β, Activin and Nodal are known to activate SMAD2/3, while BMPs and GDFs are known to activate SMAD1/5/8 signaling pathways. Activin-A binds to type II transmembrane serine threonine kinase receptor (ActRIIA or ActRIIB), which in turn activates type I receptor (ActRIB) leading to phosphorylation of SMAD2/SMAD3. Upon phosphorylation, SMAD2/3 forms a complex with SMAD4, which then translocates to nucleus. In the nucleus, SMAD2/3/4 complex, along with other co-factors regulates expression of a large number of genes. Unlike TGF-β, role of Activin in cancer is not well understood. Activin has been shown to be overexpressed in several cancers including metastatic prostate cancer, colorectal cancer, lung cancer, hepatocellular carcinoma and pancreatic cancer. Activin signaling has been shown to promote aggressiveness of esophageal squamous cell carcinoma and enhancing skin tumorigenesis and progression. Nodal, which binds to the same set of receptors, has also been shown to be overexpressed in several cancers. However, role of Activins in breast cancer progression is not well studied. Activin is expressed by normal breast epithelium and is known to play a role in mammary gland development. Earlier, a study had reported downregulation of Activin signaling in breast tumors. On the contrary, increased serum level of Activin has been reported in women with metastatic breast cancers. It is pertinent to mention here that TGF-β, which has been implicated in the progression and metastatic spread of breast cancers, also functions through the same set of downstream effectors- SMAD2 and SMAD3. Hence we wanted to evaluate the status of Activin signaling pathway in breast tumors and investigate its functional role in cancer progression. Gene expression profiling of 80 breast tumors and 20 normal samples was earlier performed in our laboratory revealed overexpression of INHBA in tumors compared to normal tissue samples. An independent set of 30 tumor and 15 normal samples were used to verify these results. Real-time PCR analysis revealed around 11.31 fold upregulation (p<0.001) of INHBA in breast tumors in comparison to normals. While no change in expression of INHA was observed, INHBB was found to be significantly downregulated in tumor samples. These results indicated upregulation of Activin-A in breast tumors. Further, a significant upregulation of ACVR2A and SMAD2 which act as signal transducers of Activin signaling pathway, was observed in breast tumors. Interestingly, while an increase in the expression of TGF-β1 was observed, TGFBR2 was found to be significantly downregulated in breast tumors. In addition, PCR analysis revealed significant downregulation of FST, β-glycan, IGSF1 and IGSF10, which act as negative regulators of Activin signaling pathway. Functional antagonism between TGF-β/Activin and BMP signaling pathway has been shown in both development and disease. Further analysis revealed that various BMPs including BMP2, BMP4 and BMP6 are downregulated in breast tumors compared to normal tissue samples. Various components and regulators of BMP signaling pathway were also found to be deregulated, indicating suppression of BMP signaling in breast tumors. To evaluate whether Activin signaling is active in breast tumor cells, immunohistochemistry with another set of 13 normal and 29 tumor samples was performed. Immunohistochemistry analysis revealed that most of the tumors have higher levels of Activin-A compared to normals tissues. Interestingly, no significant changes in expression of Activin-A was observed between normals and low grade tumors, suggesting that Activin-A may play an important role towards the late stages of the disease. In good correlation, breast tumors showed increased phospho SMAD2 and phospho SMAD3 levels compared to normal tissues. Also, in the same set of tumors, BMP2 staining showed a reduced expression pattern compared to normal tissues. Expression of inhibin in some normal and breast tumor samples revealed that most of the tumor samples have lower levels of inhibin compared to normal tissues. In order to understand the role of Activin-A in cancer progression, a panel of cell lines was selected. Treatment of cells with Activin-A resulted in activation of canonical SMAD as well as non-canonical Erk1/2 and PI3K signaling pathways. However, Activin-A treatment did not lead to activation of TAK1/p38 MAPK pathway. To begin with, it was important to evaluate effect of Activin-A on proliferation of various cell lines. Primarily, SMAD2/3 signaling pathway inhibits proliferation of normal epithelial cells, and hence, it is considered to have a tumor suppressive role. owever, this signaling pathway remains intact in most ( 98%) of the breast cancers. BrdU incorporation assay showed that Activin-A does not promote proliferation of cells under monolayer culture conditions. However, soft agar assay results showed that Activin signaling promotes anchorage independent growth of cancer cells. TGF-β is widely known as an inducer of epithelial mesenchymal transition (EMT). Also, EMT is considered to be a prerequisite for epithelial cells to undergo migration and invasion. During EMT, cells loose epithelial characteristics and acquire mesenchymal features along with cytoskeletal rearrangement. Treatment of cells with Activin-A resulted in downregulation of E-cadherin and upregulation of various mesenchymal markers. In addition, confocal microscopy imaging revealed a mesenchymal morphology of cells treated with Activin-A. Also, collagen gel contraction assay results indicated that Activin-A enhances the contractile property of HaCaT cells significantly. Cells undergone EMT are believed to acquire migratory and Invasive behaviour. In agreement with this, both scratch assay and trans-well migration assay showed that Activin-A enhances the migration of various cell lines. Further, Trans-well matrigel invasion assays were performed to assess how Activin affects invasion of various cancer cells. Matrigel invasion assay results showed that Activin-A enhances invasion of various cancer cell lines significantly. Also, RT-PCR, zymography and Luciferase assay results showed that Activin-A induces MMP2 expression. As described earlier, Activin-A activates both canonical as well as non canonical signaling pathways. In this direction, it was interesting to investigate the contribution of SMAD signaling pathway in pro-tumorigenic actions of Activin-A. Inhibiting SMAD3 activity either by its stable knockdown or by using a SMAD3 specific small molecule inhibitor revealed that Activin-A regulation of EMT markers is SMAD3 dependent. Further, it was observed that SMAD3 contributes significantly in mediating Activin-A induced migration and invasion. Hence, it is likely that SMADs may play an important role in breast tumor progression. Next, stable overexpression of Activin-A in MCF-7 or its knockdown in MDA-MB-231 and H460 cells was performed to assess the effect of Activin-A on the behaviour of these cells. BrdU assay indicated no change in proliferation of cells upon overexpression or knockdown of Activin-A. However, soft agar assay results showed that Activin-A expression affects anchorage independent growth of these cells. MCF-7 cells are generally considered to be less aggressive in their tumor forming ability. Activin-A overexpressing MCF7 cells and control cells were respectively injected into right and left flank of immunocompromised mice and followed till the tumors reached to a prominent size. Our results show that Activin-A overexpressing MCF-7 cells have better tumor forming ability in comparison to control cells. In contrast to MCF-7 cells, MDA-MB-231 cells are known to be aggressive in their tumorigenic potential. In order to understand the effect of Activin-A knockdown on the tumor forming ability in MDA-MB-231 cells, 0.5 million cells (optimal cell number generally used is 1-2 million) were injected subcutaneously in immunocompromised mice. The results showed that while control cells gave rise to a tumor in 7 out of 10 animals, Activin-A knockdown cells could form a tumor in only 3 out of 10 animals. Also, the tumors formed by control cells were significantly larger by weight as compared to tumors formed by knockdown cells. Further, immunohistochemistry showed that tumors formed by MCF-7 cells overexpressing Activin-A have higher Ki-67 percentage as compared to control tumors. One of the factors known to be important for tumor growth is VEGF, which leads to recruitment of blood vessels and hence providing nourishment to the tumor cells. Hence Activin-A regulation of VEGF expression was evaluated next. Activin-A treatment or its stable overexpression in MCF-7 cells resulted in increased VEGF expression in these cells. This was also confirmed by VEGF promoter activity assay. To assess if Activin-A can play a role in metastatic spread of cancer cells, tail vein injection of Activin-A overexpressing MCF-7 cells was performed in immunocompromised mice. Even though no significant difference was found in the number of nodules formed by control or Activin-A overexpressing cells, it was observed that Activin-A overexpressing cells formed much bigger nodules as compared to the control cells. This suggests that Activin-A may play an important part in the establishment of metastases from the disseminated cancer cells. Tumor forming ability of cancer cells and aggressiveness of various cancers has been associated with the presence of cells having stem-like phenotype. In this direction, CD44high and CD24low expression status was analysed upon overexpression and knockdown of Activin-A in MCF-7 and MDA-MB-231 cells respectively. FACS analysis of Activin-A overexpressing MCF-7 cells and Activin-A knockdown MDA-MB-231 cells shows that Activin-A expression leads to enrichment of breast cancer stem-like cells. In conclusion, this study highlights the importance of Activin-A signaling pathway in the progression of breast tumors. It is also important to note the role of SMAD signalling in the progression of breast cancers since these effectors are common between TGF-β, Activin and nodal factors, which have been shown to be involved in cancer progression in a context dependent manner.

No description available.

Le fer, un métal de transition, est requis pour la survie de presque tout les organismes vivant à cause de son habilité à accepter ou donner un électron et donc à catalyser plusieurs réactions biochimique fondamentales. Cependant, la même propriété permet aussi au fer ionique d’accélérer la formation de radicaux libres et donc le fer peut potentiellement avoir des effets néfastes. Conséquemment, l’homéostasie du fer doit être étroitement régulé, tant au niveau cellulaire que systémique. Notre étude met l’emphase sur deux molécules importante pour régulation du métabolisme du fer : la lipocaline 2 (Lcn2) et l’hepcidine. Lcn2, une protéine de phase aiguë, est impliquée dans le transport du fer par les sidérophores. Lcn2 est un candidat potentiel comme transporteur du fer qui pourrait être responsable de l’accumulation excessive du fer non lié à la transferrine dans le foie des patients atteints d’hémochromatose héréditaire (HH). Nous avons généré des souris double-déficiente HfeLcn2 pour évaluer l’importance de Lcn2 dans la pathogenèse de surcharge en fer hépatique dans les souris knock-out Hfe (Hfe -/-). Notre étude révèle que la délétion de Lcn2 dans les souris Hfe-/- n’influence pas leur accumulation de fer hépatique ou leur réponse à une surcharge en fer. Le phénotype des souries HfeLcn2-/- demeure indiscernable de celui des souris Hfe-/-. Nos données impliquent que Lcn2 n’est pas essentiel pour la livraison du fer aux hépatocytes dans l’HH. L’hepcidine, un régulateur clé du métabolisme du fer, est un petit peptide antimicrobien produit par le foie et qui régule l’absorption intestinale du fer et son recyclage par les macrophages. L’expression de l’hepcidine est induite par la surcharge en fer et l’inflammation, tandis que, à l'inverse, elle est inhibée par l'anémie et l'hypoxie. Dans certaine situations pathologique, l’hepcidine est régulée dans des directions opposées par plus d’un régulateur. Nous avons, en outre, analysé comment les différents facteurs influencent l’expression de l’hepcidine in vivo en utilisant un modèle de souris avec un métabolisme du fer altéré. Nous avons examiné la régulation de l’hepcidine en présence de stimuli opposés, ainsi que la contribution des médiateurs et des voix de signalisation en aval de l’expression de l’hepcidine. Nous avons démontré que l'érythropoïèse, lorsque stimulé par l’érythropoïétine, mais pas par l’hypoxie, diminue l’expression de l’hepcidine d’une façon dépendante de la dose, même en présence de lipopolysaccharides ou de surcharge de fer alimentaire, qui peuvent agir de manière additive. De plus, l’entraînement érythropoïétique inhibe tant la voix inflammatoire que celle de détection du fer, du moins en partie, par la suppression du signal IL-6/STAT3 et BMP/SMAD4 in vivo. Au total, nos données suggèrent que le niveau d’expression de l’hepcidine en présence de signaux opposés est déterminé par la force du stimulus individuel plutôt que par une hiérarchie absolue. Ces découvertes sont pertinentes pour le traitement de l’anémie des maladies chronique et les désordres de surcharge en fer. / Iron, a transition metal, is required for survival by almost all living organisms due to its ability to accept or donate electrons and thus to catalyze many fundamental biochemical reactions. However, the same properties also allow ionic iron to accelerate the formation of free radicals and as such iron has the potential for deleterious effects. Consequently, iron homeostasis must be tightly regulated at both cellular and systemic levels. Our studies focused on two important molecules in the regulation of iron metabolism, namely, lipocalin 2 (Lcn2) and hepcidin. Lcn2, an acute phase protein, is involved in iron trafficking via siderophores. Lcn2 has emerged as a candidate iron-transporter that may be responsible for excessive non-transferrin-bound iron (NTBI) accumulation in the liver of hereditary hemochromatosis (HH) patients. We generated HfeLcn2 double-deficient mice to evaluate the importance of Lcn2 in the pathogenesis of hepatic iron loading in Hfe knockout mice. Our studies revealed that deletion of Lcn2 in Hfe-knockout mice does not influence hepatic iron accumulation in Hfe-/- mice, or their response to iron loading, as the phenotype of HfeLcn2-/- mice remained indistinguishable from that of Hfe-/- mice. Our data imply that Lcn2 is not essential for iron delivery to hepatocytes in HH. Hepcidin, a key regulator of iron metabolism, is a small antimicrobial peptide produced by the liver that regulates intestinal iron absorption and iron recycling by macrophages. Hepcidin expression is induced by iron-loading and inflammation while, conversely, being inhibited by anemia and hypoxia. Under certain pathologic situations, hepcidin is regulated in opposite directions by more than one regulator. We further investigated how different factors influence hepcidin expression in vivo using mouse models of altered iron metabolism. We examined hepcidin regulation in the presence of opposing stimuli as well as the contributions of mediators and downstream signaling pathways of hepcidin expression. We show that erythropoiesis drive, when stimulated by erythropoietin but not by hypoxia, down-regulates hepcidin in a dose-dependent manner, even in the presence of lipopolysaccharide or dietary iron-loading, which may act additively. Moreover, erythropoietic drive inhibited both the inflammatory and iron-sensing pathways, at least in part, via the suppression of IL-6/STAT3 and BMP/SMAD4 signaling in vivo. Altogether, our data suggest that hepcidin expression levels in the presence of opposing signaling are determined by the strength of the individual stimuli rather than by an absolute hierarchy. These findings are pertinent for the treatment of the anemia of chronic disease and iron-loading disorders.

Tang, Yongjiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 159-202). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

<p>Thyroid growth and function is partly regulated by growth factors binding to receptors on the cell surface. In the present thesis, the transforming growth factor-β (TGF-β) superfamily members have been studied for their role in regulation of growth and differentiation of both normal and neoplastic thyroid epithelial cells.</p><p>TGF-β1 is a negative regulator of thyrocyte growth and function. However, the importance of other TGF-β superfamily members has not been fully investigated. TGF-β1, activin A, bone morphogenetic protein (BMP)-7 and their receptors were found to be expressed in porcine thyrocytes. In addition to TGF-β1, activin A was also found to be a negative regulator of thyroid growth and function, and both stimulated phosphorylation and nuclear translocation of Smad proteins. Furthermore, TGF-β1 and epidermal growth factor (EGF) demonstrated a synergistic negative effect on thyrocyte differentiation. Simultaneous addition of the two factors resulted in a loss of the transepithelial resistance and expression of the epithelial marker E-cadherin. This was followed by a transient expression of N-cadherin.</p><p>Despite the extremely malignant character of anaplastic thyroid carcinoma (ATC) tumor cells, established cell lines are still responsive to TGF-β1. A majority of the cell lines were also found to be growth inhibited by BMP-7. BMP-7 induced cell cycle arrest of the ATC cell line HTh 74 in a dose- and cell density-dependent manner. This was associated with upregulation of p21^CIP1 and p27^KIP1, decreased cyclin-dependent kinase (Cdk) activity and hypophosphorylation of the retinoblastoma protein (pRb). TGF-β1, and to some extent also BMP-7, induced the expression of N-cadherin and matrix metalloproteinase (MMP)-2 and -9. Stimulation of HTh 74 cells with TGF-β1 increased the migration through a reconstituted basement membrane indicating an increased invasive phenotype of the cells.</p><p>Taken together, these data show that TGF-β superfamily members not only affect growth and function of normal thyroid follicle cells but may also, in combination with EGF, play a role in cell dedifferentiation. This study additionally suggests that the TGF-β superfamily members may be important for the invasive properties of ATC cells.</p>

<p>The transforming growth factor-β (TGF-β) superfamily consists of related multifunctional cytokines, which include TGF-βs, activins, and bone morphogenetic proteins (BMPs) and coordinate several biological responses in diverse cell types. The biological activity of TGF-β members is executed by transmembrane serine/threonine kinase receptors and intracellular Smad proteins. The effects of TGF-β on the epithelium are of high interest. Carcinomas (tumors of epithelial origin) are the most common type of human cancer and frequently exhibit aberrant responses to TGF-β. Therefore, TGF-β can be defined as tumor suppressor as it inhibits growth of normal epithelial cells. However, TGF-β also induces an epithelial-mesenchymal transition (EMT), a key component of metastasis, and thus promotes cancer spread.</p><p>The scope of this thesis is the mechanism of TGF-β signaling in epithelial cells. We established that only TGF-β, but not BMP pathways can elicit EMT. Moreover, we found that Smad signaling is critical for regulation of EMT. In a transcriptomic analysis, we identified a large group of novel genes, whose regulation is pivotal for TGF-β-induced EMT and metastasis. We focused on two of such genes, <i>Id2</i> and <i>Id3</i>. Interestingly, we found that TGF-β-induced repression of <i>Ids</i> is necessary for inducing EMT and potent cell cycle arrest. BMP increases expression of <i>Ids</i> and therefore it cannot induce the same biological responses as TGF-β. Hence, knock-down of endogenous Id2 and Id3 proteins sensitized epithelial cell to BMP-7. We proposed a model, in which Id2 and Id3 are important components controlling concerted regulation of cell proliferation and EMT downstream of TGF-β pathways.</p><p>Furthermore, we identified a serine/threonine kinase, <i>SNF1LK</i>, whose mRNA is rapidly induced by TGF-β in epithelial cells. We found that SNF1LK is a negative regulator of the TGF-β pathway and it promotes TGF-β receptor turnover. Subsequently, we demonstrated that SNF1LK together with Smad7 and Smurf2 targets TGF-β receptor for ubiquitin-dependent degradation. Furthermore, SNF1LK interacts with proteasomes, suggesting that SNF1LK serves as bridge between ubiquitinated receptors and proteasomes, helping proteasomes to recognize the ubiquitinated cargo destined for degradation. We therefore established a novel negative feedback regulatory mechanism of TGF-β signaling. </p>