Doctor of Philosophy (PhD) / Background: The development of the blood supply in the primate retina has been extensively studied; however the relationship of the differentiating retina to the choroidal blood supply is less well known. The interaction of astrocytes and vascular endothelial cells promotes the development of the retinal vasculature from 14 weeks’ gestation (WG). Initially, astrocytes lead the developing capillaries from the optic nerve towards the macular area. However, neither astrocytes nor endothelial cells enter a prescribed avascular area, within which the fovea later forms. This may be attributed to expression of a factor that inhibits astrocyte and endothelial cell proliferation in the fovea. A factor found in the CNS that is already known to have these effects is transforming growth factor-β (TGF-β). Aims: This thesis investigated the relationship between retinal maturation and choroidal blood vessel supply and the possible role for TGF-β as an antiangiogenic factor in maintaining an avascular fovea during primate retinal development. Methods: Human eyes between 11 WG and 40 years were obtained with ethical approval from Prince of Wales Hospital and the NSW Lions Eye Bank and fixed and sectioned for histological procedures or prepared for polymerase chain reaction (PCR). Macaque eyes from foetal day (fd) 64 to postnatal 11years (p11y) were obtained from Bogor Agriculture University, Indonesia with the approval of the Ethics Committee of the University of Washington, Seattle, USA. Macaque eyes were also fixed and sectioned for immunohistochemistry and in situ hybridisation. RNA was extracted from human foetal retinas and used for RTPCR (Reverse Transcriptase PCR), QPCR (Quantitative PCR) and preparation of riboprobes. PCR products were analysed using both restriction digest and sequencing. RTPCR was used to identify TGF-β1, TGF-β2 and TGF-β3 in the developing human and in the developing and adult macaque retinas whilst QPCR was used to quantify the TGF-β isoforms in central compared to peripheral retina and in foetal compared to adult retina. In situ hybridisation was performed according to a standard protocol and visualised using Roche HNPP Fast Red detection set with designed riboprobes for TGF-β1, TGF-β2 and TGF-β3 (DIG RNA labelling kit). Some sections were counterstained with vimentin antibody. Immunohistochemistry was performed on human retina and choroid sections using antibodies to CD34 and Ki67 and on human and macaque retina using antibodies to synaptophysin, vimentin, GFAP, calbindin, S-opsin, RG-opsin, rhodopsin, TGF-β1, TGF-β2, TGF-β3 and their receptors TβRI and TβRII. Sections of the retina were imaged and analysed using either a Leica Confocal microscope and TCSNT software or Zeiss Confocal microscope and LSM 5 Pascal software. Data from the human retina and choroid sections corresponding to different regions (foveal, parafoveal nasal, parafoveal temporal, nasal and temporal) was collected to measure the number of Ki-67 immunolabelled mitotic endothelial cells and the area of CD34 immunolabelled choriocapillaris using Adobe Photoshop version 5.0.2, NIH software version 1.62 (measurement macros) and Excel. In the human and macaque sections the intensity of TGF-β protein and mRNA expression was captured from different regions of the retina (foveal, parafoveal nasal, parafoveal temporal, nasal, temporal, nasal to disc) to compile montages. Montages were then re-imported into LSM 5 Pascal software to measure the optical density across each montage along the ganglion cell layer, outer neuroblastic zone and photoreceptor layer collecting data in Excel for graphical representation. In addition to the montages, individual sections were assessed for co-localisation of TGF-β and TβR to various retinal cell types. Results: Analyses of choriocapillaris area and endothelial cell (EC) proliferation were able to demonstrate that the area of choriocapillaris endothelium is greater in the foveal region at all ages (14-18.5WG), that the rate of choriocapillaris EC proliferation declines dramatically over this same period and that the lowest rates of EC proliferation are at the incipient fovea. Most importantly these findings indicate that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons which are more developed with higher oxygen and nutrient demands, which is the mechanism widely thought to regulate development of the retinal vasculature. PCR showed all TGF-β isoforms to be present in the human developing and adult retina. QPCR revealed that TGF-β2 was the most predominant isoform, followed by TGF-β3 with very small amounts of TGF-β1 seen. The isoforms were more abundant in developing rather than adult retina and in central rather than peripheral retina. Studies of the distribution of TGF-β protein and mRNA using immunohistochemistry and in situ hybridisation confirmed the low levels of TGF-β1 protein and mRNA observed in QPCR and demonstrated distinct centroperipheral gradients in the photoreceptor layer for TGF-β2 and TGF-β3. Relative high amounts of TGF-β in the fovea could affect vascular patterning due to TβRI seen in astrocytes which lead the blood vessels at the foveal rim at the level of the ganglion cell plexus. TGF-β2 and TGF-β3 expression is detected before formation of the foveal avascular zone (FAZ) at fd64 (~15WG) - fd73 (~17WG) with levels peaking in the foveal region at fd105 (~25WG) by the time the FAZ forms. Conclusions: This thesis has shown that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons as reduced rates of EC proliferation in the ‘foveal’ chorioretinal location were observed at all ages studied between 14 and 18.5WG. The findings suggest that mechanisms regulating proliferation and growth of the choroidal vasculature are independent of differentiation in the neural retina and are therefore different to those governing the formation of the retinal vasculature. All TGF-β isoforms are expressed in developing and adult human and macaque retina with TGF-β2 being the predominant isoform. TGF-β isoforms are more abundant in central compared to peripheral retina and in developing compared to adult retina. Centro-peripheral gradients of TGF-β2 and TGF-β3 across the photoreceptor layer and TβRI on astrocytes support the presence of TGF-β in the fovea as an antiproliferative and antiangiogenic factor by helping to define the FAZ early in development, well before 23-25 WG in humans and before fd100 in macaques.

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.

TGF-β negatively affects the maintenance and expansion of hematopoietic stem cells ex vivo and its inhibition has been widely studied as a treatment for numerous hematopoietic disorders and cancers. Current inhibitory strategies (small molecule ATP competitors and neutralizing antibodies) are compared to a novel cell-permeable peptide-based inhibitor of TGF-β RI. Multiple levels of assay from biochemical to functional are utilized with the aim of applying the most successful inhibitor to hematopoietic stem cell culture. The neutralizing antibody proved ineffective in the short-term biochemical assay but was extremely effective at neutralizing TGF-β signalling in proliferation and hematopoietic colony-forming cell assays with no evidence of toxicity. The small molecule inhibitors (SD-208 and Pyrazole TGF-β RI inhibitor) were equally effective at micromolar levels in all forms of assay, with SD-208 being slightly more potent. The novel peptide inhibitor proved ineffective in all assays, which is likely a result of its rapid degradation in live cells.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor. Little is known about aggressive forms of this disease. In order to identify pathways mediating aggressiveness in MB, we performed microarray experiments. Primary human MBs were compared to their patient matched recurrent or metastatic counterparts. Murine tumors from two MB mouse models that present with differing clinical severities were also evaluated. We identified the Transforming Growth Factor-beta (TGF-β) as a potential contributor to MB pathogenesis in both species. Smad3, a major downstream component of the TGF-β pathway, was shown to correlate with MB metastasis and survival in human tissue. Similarly, Smad3 expression during development identified a subset of cerebellar neuronal precursors as putative cells of origin for the Smad3 positive MBs. To our knowledge, this is the first study that links TGF-β to MB pathogenesis. Our research suggests that canonical activation of this pathway leads to better prognosis for patients.

Medulloblastoma (MB) is the most common pediatric malignant brain tumor. Little is known about aggressive forms of this disease. In order to identify pathways mediating aggressiveness in MB, we performed microarray experiments. Primary human MBs were compared to their patient matched recurrent or metastatic counterparts. Murine tumors from two MB mouse models that present with differing clinical severities were also evaluated. We identified the Transforming Growth Factor-beta (TGF-β) as a potential contributor to MB pathogenesis in both species. Smad3, a major downstream component of the TGF-β pathway, was shown to correlate with MB metastasis and survival in human tissue. Similarly, Smad3 expression during development identified a subset of cerebellar neuronal precursors as putative cells of origin for the Smad3 positive MBs. To our knowledge, this is the first study that links TGF-β to MB pathogenesis. Our research suggests that canonical activation of this pathway leads to better prognosis for patients.

TGF-β negatively affects the maintenance and expansion of hematopoietic stem cells ex vivo and its inhibition has been widely studied as a treatment for numerous hematopoietic disorders and cancers. Current inhibitory strategies (small molecule ATP competitors and neutralizing antibodies) are compared to a novel cell-permeable peptide-based inhibitor of TGF-β RI. Multiple levels of assay from biochemical to functional are utilized with the aim of applying the most successful inhibitor to hematopoietic stem cell culture. The neutralizing antibody proved ineffective in the short-term biochemical assay but was extremely effective at neutralizing TGF-β signalling in proliferation and hematopoietic colony-forming cell assays with no evidence of toxicity. The small molecule inhibitors (SD-208 and Pyrazole TGF-β RI inhibitor) were equally effective at micromolar levels in all forms of assay, with SD-208 being slightly more potent. The novel peptide inhibitor proved ineffective in all assays, which is likely a result of its rapid degradation in live cells.

INTRODUCCIÓ: En les patologies articulars que afecten el cartílag existeix una relació bidireccional entre la degradació del teixit i la resposta inflamatòria. El plasma ric en plaquetes conté factors moduladors de la inflamació i promotors de la regeneració. Aquests factors podrien actuar sobre els condròcits modificant-ne l’expressió de citocines i molècules de la matriu cartilaginosa, de forma que es generaria un entorn favorable per la recuperació del dany. OBJECTIUS: Establir models in vitro de condròcits tractats amb LPS i de condròcits tractats amb agents oxidants i avaluar l’efecte del plasma ric en plaquetes sobre aquests models cel·lulars. MATERIAL I MÈTODES: S’han establert cultius primaris de condròcits a partir de biòpsies de cartílag articular i s’han tractat amb LPS (100 μg/ml) o amb H2O2 (1mM) durant 24 h. S’ha valorat l’activitat metabòlica dels condròcits mitjançant la quantificació de glicosaminoglicans (GAGs) en els sobrenadants dels cultius i també s’ha valorat el patró de citocines i factors de creixement (IL-12p70, TNF-_, IL-10, IL-6, IL-1β, IL-8 i TGF-β) alliberats pels condròcits en el sobrenadant, mitjançant citometria de flux o ELISA. S’ha generat plasma ric en plaquetes a partir de sang sencera i se n’ha valorat el contingut en TGF-β. S’han tractat els cultius de condròcits amb PRP (25% i 50%) i s’ha valorat l’activitat metabòlica i el perfil de citocines dels condròcits. RESULTATS: El tractament dels condròcits amb LPS provoca un increment de GAGs i de les citocines TNF-α, IL-6, IL-8 i IL-10 en els sobrenadant, mentre que disminueixen els nivells de TGF-β. A l’afegir PRP ric en TGF-β al cultiu, el TNF-α i la IL-10 del sobrenadant disminueixen. L’oxidació dels condròcits amb H2O2 provoca la disminució de GAGs en el sobrenadant però no altera el perfil de citocines. L’efecte del PRP sobre el cultiu de condròcits en condicions d’oxidació genera un increment de la IL-8 i un lleuger augment de la IL-6. CONCLUSIONS: Els condròcits participen de forma activa en la resposta inflamatòria ja que quan estan en presència d’un agent com el LPS canvien el perfil de citocines inflamatòries i reguladores que sintetitzen. El PRP, ric en TGF-β, modifica el patró de citocines cap a un perfil menys inflamatori i pot promoure la proliferació dels condròcits. El tractament amb PRP de condròcits en un entorn oxidatiu provoca uns efectes similars als del tractament amb PRP sobre cultius normals, però en menor intensitat. / INTRODUCCIÓN:En las patologías articulares que afectan el cartílago existe una relación bidireccional entre la degradación del tejido y la respuesta inflamatoria, de manera que se generan situaciones de inflamación crónica y de perpetuación de la degradación del tejido. El plasma rico en plaquetas (PRP) contiene factores moduladores de la inflamación y promotores de la regeneración como el TGF-β. La hipótesis de este trabajo es que estos factores podrían actuar sobre los condrocitos modificando la expresión de citocinas y de moléculas de la matriz cartilaginosa, de forma que se generaría un entorno favorable para la recuperación del daño. OBJETIVOS: Establecer modelos in vitro de condrocitos tratados con LPS y de condrocitos tratados con agentes oxidantes, y evaluar el efecto del plasma rico en plaquetas (PRP) en estos modelos de daño celular. MATERIALES Y MÉTODOS: Se han establecido cultivos primarios de condrocitos a partir de biopsias de cartílago articular procedentes de pacientes (70-90 años) sometidos a cirugía de reemplazamiento. Se han generado modelos de daño celular tratando cultivos de condrocitos con LPS (100 µg/ml) o con H2O2 (1mM) durante 24 h. Se ha valorado la actividad sintética de los condrocitos con la cuantificación de los glucosaminoglicanos (GAGs) en los sobrenadantes de los cultivos mediante el ensayo colorimétrico DMMB. También se ha valorado el patrón de citocinas inflamatorias y antiinflamatorias, y de factores de crecimiento (IL-12p70, TNF-α, IL-10, IL-6, IL-1β, IL-8 y TGF-β) liberados por los condrocitos en el sobrenadante, mediante citometría de flujo (ensayo CBA) o mediante ELISA. Se ha generado plasma rico en plaquetas mediante centrifugación (460 g) de sangre entera y se ha determinado el contenido de TGF-β después de la activación de las plaquetas con 45mM CaCl2. Se han tratado los diferentes cultivos de condrocitos con PRP (25% y 50%) y se ha valorado el efecto de éste sobre la actividad sintética y el perfil de citocinas de los condrocitos. RESULTADOS:El tratamiento de los condrocitos con LPS provoca un incremento de GAGs y de las citocinas TNF-α, IL-6, IL-8 e IL-10 en los sobrenadantes, mientras que los niveles de TGF-β disminuyen. Cuando se añade PRP rico en TGF-β al cultivo, la cantidad de TNF-α y de IL-10 en el sobrenadante disminuye, mientras que la cantidad de IL-6 e IL-8 se mantiene elevada, como también se mantiene la capacidad de síntesis de GAGs. La oxidación de los condrocitos con H2O2 provoca la disminución de GAGs en el sobrenadante, pero no modifica el perfil de citocinas. El efecto del PRP sobre el cultivo de condrocitos en condiciones de oxidación recupera la síntesis de GAGs y genera un incremento de la IL-8 y un ligero aumento de la IL-6. CONCLUSIONES: Los condrocitos participan de forma activa en la respuesta inflamatoria ya que, cuando están en presencia de un agente como el LPS, cambian el perfil de citocinas inflamatorias y reguladoras que sintetizan. Con la adición en el cultivo de PRP, rico en TGF-β, se estaría supliendo la disminución de TGF-β provocada por el LPS en los condrocitos y provocaría el cambio en el patrón de citocinas hacia un perfil menos inflamatorio. Si una situación similar sucediera in vivo, con el uso del PRP, se estaría generando un entorno favorable para la recuperación del daño. El tratamiento in vitro con PRP de condrocitos en un entorno oxidativo, produce efectos similares a los tratamientos con PRP sobre cultivos normales, pero en menor intensidad, pudiendo, eso sí, recuperar la actividad sintética de matriz extracelular. / INTRODUCCIÓN:En las patologías articulares que afectan el cartílago existe una relación bidireccional entre la degradación del tejido y la respuesta inflamatoria, de manera que se generan situaciones de inflamación crónica y de perpetuación de la degradación del tejido. El plasma rico en plaquetas (PRP) contiene factores moduladores de la inflamación y promotores de la regeneración como el TGF-β. La hipótesis de este trabajo es que estos factores podrían actuar sobre los condrocitos modificando la expresión de citocinas y de moléculas de la matriz cartilaginosa, de forma que se generaría un entorno favorable para la recuperación del daño. OBJETIVOS: Establecer modelos in vitro de condrocitos tratados con LPS y de condrocitos tratados con agentes oxidantes, y evaluar el efecto del plasma rico en plaquetas (PRP) en estos modelos de daño celular. MATERIALES Y MÉTODOS: Se han establecido cultivos primarios de condrocitos a partir de biopsias de cartílago articular procedentes de pacientes (70-90 años) sometidos a cirugía de reemplazamiento. Se han generado modelos de daño celular tratando cultivos de condrocitos con LPS (100 µg/ml) o con H2O2 (1mM) durante 24 h. Se ha valorado la actividad sintética de los condrocitos con la cuantificación de los glucosaminoglicanos (GAGs) en los sobrenadantes de los cultivos mediante el ensayo colorimétrico DMMB. También se ha valorado el patrón de citocinas inflamatorias y antiinflamatorias, y de factores de crecimiento (IL-12p70, TNF-α, IL-10, IL-6, IL-1β, IL-8 y TGF-β) liberados por los condrocitos en el sobrenadante, mediante citometría de flujo (ensayo CBA) o mediante ELISA. Se ha generado plasma rico en plaquetas mediante centrifugación (460 g) de sangre entera y se ha determinado el contenido de TGF-β después de la activación de las plaquetas con 45mM CaCl2. Se han tratado los diferentes cultivos de condrocitos con PRP (25% y 50%) y se ha valorado el efecto de éste sobre la actividad sintética y el perfil de citocinas de los condrocitos. RESULTADOS:El tratamiento de los condrocitos con LPS provoca un incremento de GAGs y de las citocinas TNF-α, IL-6, IL-8 e IL-10 en los sobrenadantes, mientras que los niveles de TGF-β disminuyen. Cuando se añade PRP rico en TGF-β al cultivo, la cantidad de TNF-α y de IL-10 en el sobrenadante disminuye, mientras que la cantidad de IL-6 e IL-8 se mantiene elevada, como también se mantiene la capacidad de síntesis de GAGs. La oxidación de los condrocitos con H2O2 provoca la disminución de GAGs en el sobrenadante, pero no modifica el perfil de citocinas. El efecto del PRP sobre el cultivo de condrocitos en condiciones de oxidación recupera la síntesis de GAGs y genera un incremento de la IL-8 y un ligero aumento de la IL-6. CONCLUSIONES: Los condrocitos participan de forma activa en la respuesta inflamatoria ya que, cuando están en presencia de un agente como el LPS, cambian el perfil de citocinas inflamatorias y reguladoras que sintetizan. Con la adición en el cultivo de PRP, rico en TGF-β, se estaría supliendo la disminución de TGF-β provocada por el LPS en los condrocitos y provocaría el cambio en el patrón de citocinas hacia un perfil menos inflamatorio. Si una situación similar sucediera in vivo, con el uso del PRP, se estaría generando un entorno favorable para la recuperación del daño. El tratamiento in vitro con PRP de condrocitos en un entorno oxidativo, produce efectos similares a los tratamientos con PRP sobre cultivos normales, pero en menor intensidad, pudiendo, eso sí, recuperar la actividad sintética de matriz extracelular.

En los últimos años nuestro grupo ha estudiado las diferentes vías de señalización inducidas por TGF-β en hepatocitos fetales de rata. A dosis bajas, el TGF-β inhibe el crecimiento, pero a concentraciones más elevadas es capaz de inducir apoptosis (Sanchez et al. 1995; Sanchez et al. 1996). El proceso apoptótico está mediado por un incremento en el contenido intracelular de ROS, dependiente de la síntesis de novo de proteínas, y que correlaciona con una caída en los niveles intracelulares de glutatión (Sanchez et al. 1997). La muerte inducida por TGF-β en estas células se correlaciona con un descenso en los niveles de Bcl-xL, la despolarización de la membrana mitocondrial, la salida de citocromo c y posterior activación de caspasas (Herrera et al. 2001a; Herrera et al. 2001b). El incremento de ROS intracelulares se produce por activación de un sistema NADPH oxidasa y por disminución en la expresión de proteínas antioxidantes (Herrera et al. 2004). Recientemente hemos descrito que la NADPH oxidasa NOX4 se induce en condiciones pro-apoptóticas (Carmona-Cuenca et al. 2006), pero otras NADPH oxidasas podrían jugar un papel diferente en la señalización inducida por TGF-β (Murillo et al., 2007). La muerte inducida por TGF-β puede ser inhibida por EGF a través de la activación de PI3K (Fabregat et al. 2000), que contrarresta la expresión de Nox4 (Carmona-Cuenca et al. 2006). Sin embargo, el 40-50% de las células sobreviven a los efectos apoptóticos del TGF-β y adquieren una morfología fibroblastoide (Sanchez et al. 1999). Esto es debido a que el TGF-β también induce señales anti-apoptóticas en los hepatocitos fetales, proceso que requiere la activación del EGFR, producida por un aumento en los niveles de expresión de sus ligandos y activación de la metaloproteasa TACE/ADAM17 que los proteoliza y activa (Valdes et al. 2004; Murillo et al. 2005; Del Castillo et al. 2006; Murillo et al. 2007). Las células que sobreviven al TGF-β responden a esta citoquina en términos de migración e invasión, disminuyendo la expresión de marcadores hepáticos (Sanchez et al. 1999), e induciendo un proceso de EMT (Valdes et al. 2002). La población mesenquimática resultante es resistente a la muerte inducida por TGF-β, ha sufrido un proceso de desdiferenciación y expresa marcadores de célula madre (Del Castillo et al. 2006; del Castillo et al. 2008). Esta población puede rediferenciarse tanto a un linaje hepatocítico como hacia células biliares cuando se mantienen con los medios de diferenciación adecuados. Por último, resultados preliminares al inicio de esta tesis doctoral proponían que la doble respuesta al TGF-β observada en hepatocitos fetales de rata en cultivo primario era exclusiva de este estadio del desarrollo hepático, ya que en hepatocitos adultos de rata el TGF-β sólo inducía apoptosis. La incapacidad del TGF-β de inducir señales de supervivencia parece deberse a la baja expresión de AKT y de TACE observada en hepatocitos adultos. Además, el TGF-β era incapaz de inducir un proceso de EMT en hepatocitos adultos de rata. A la vista de estos resultados se consideró de gran importancia analizar cuál podría ser la respuesta al TGF-β en células tumorales hepáticas. Así, nuestro principal objetivo en esta tesis ha sido analizar si las células de carcinoma hepatocelular responden a la muerte celular inducida por el TGF-β, y en el caso de que hayan adquirido resistencia, estudiar los mecanismos moleculares que la confieren. También queríamos saber si el TGF-β induce señales de supervivencia y un proceso de EMT en células tumorales hepáticas, y la relevancia de este proceso en la progresión del tumor hepático. Aunque quisimos iniciar el estudio con células de hepatoma de rata, debido a nuestra experiencia en este modelo de celular, consideramos muy importante también analizar la situación en células tumorales de hígado humano, ya que es conocido que los niveles de TGF-β son elevados en carcinoma hepatocelular (HCC) y diferentes evidencias han sugerido que la respuesta al TGF-β está alterada en células de HCC. / In the last years our research has focused on analyzing the signaling pathways induced by TGF-β in liver tumor cell lines, to understand the molecular mechanisms that confer resistance to its suppressor effects. TGF-β induces apoptosis in human fetal hepatocytes and in some liver tumor cells (FaO rat hepatoma, Hep3B and PLC/PRF/5 human hepatocarcinoma cells), which requires reactive oxygen species (ROS) production and up-regulation of the NADPH oxidase NOX4. This process is coincident with an increased expression of pro-apoptotic BCL-2 family members, such as BMF or BIM. However, in these same cells, TGF-β also induces anti-apoptotic signals, mediated by the activation of the epidermal growth factor receptor (EGFR) and coincident with up-regulation of the anti-apoptotic proteins BCL-XL, MCL1 or HIAP1. Inhibition of the EGFR, either by pharmacological inhibitors or through targeting knock-down with specific siRNA, significantly enhances the apoptotic response, which indicates that the EGFR plays a relevant role in conferring resistance to TGF-β-induced cell death. However, even when the EGFR is inhibited, some hepatocellular carcinoma cells, such as HepG2 or SK-Hep1, continue showing resistance to TGF-β-induced cell death. HepG2 cells are sensitized to TGF-β-induced apoptosis through the inhibition of the MEK pathway. MEK inhibition allows TGF-β to induce its pro-apoptotic program in these cells, which is coincident with NOX4 upregulation, modulation of the expression of BCL-2 family members and caspase-3 activation. It is worthy to note that activation of survival pathways, such as EGFR or MEK/ERK, in liver tumor cells confers resistance to TGF-β-induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis. Finally, our results have indicated that TGF-β is able to induce an epithelial to mesenchymal transition (EMT) process in human fetal hepatocytes, FaO rat hepatoma cells and Hep3B human hepatocarcinoma cells. TGF-β induces Snail expression, coincident with a decrease in E-cadherin mRNA and protein levels. Furthermore, cells show an increased expression of mesenchymal genes and reorganization of the actin cytoskeleton in stress fibers. Interestingly, these cells show loss of expression of specific hepatic markers and increased expression of stem cell markers. Indeed, chronic treatment with TGF-β selects a population of mesenchymal cells with a de-differentiated phenotype, reminiscent of progenitor-like cells. In summary, TGF-β induces different signals in liver tumor cells, some of them might contribute to tumor suppression (apoptosis), but others should mediate liver tumor progression and invasion.

<p>Transforming growth factor–β (TGF–β) signaling regulates a range of processes in a variety of cell types. Consequently, TGF–β plays a complex role in the progression of several types of cancers; it acts as a tumor suppressor in normal cells and early in tumor progression, yet it can promote tumor progression in later stages of cancer.</p><p>Among the cancers that TGF–β has been implicated in is glioblastoma multiforme (GBM), the most common primary brain neoplasm and one of the most lethal types of cancer. Because of its high mortality rate and the lack of effective treatments, discovering the molecular mechanisms that underlie GBM formation and growth is of great clinical interest. To this end, we investigated the function of a TGF–β target gene — the putative tumor suppressor N‐Myc downstream‐regulated gene 4 (NDRG4) — in GBM cell viability, proliferation and tumor formation. Contrary to the established roles of other NDRG family members, we found that NDRG4 expression is elevated in GBM and that NDRG4 is required for the survival of established GBM cell lines and primary GBM xenograft cells enriched for highly tumorigenic GBM cancer stem cells. Knockdown of NDRG4 expression results in G₁ cell cycle arrest followed by apoptosis that is associated with a decrease in the expression of XIAP and survivin. Finally, knockdown of NDRG4 expression in established GBM cell lines and GBM cancer stem cells results in decreased tumorigenicity following intracranial implantation of these cells into immunocompromised mice. Collectively, these data indicate that NDRG4 does not function as a tumor suppressor like other NDRG family members, but rather it is essential for GBM tumorigenicity and may represent a potential therapeutic target for this devastating disease.</p><p>In the second portion of this dissertation, we examine the TGF–β cytostatic signaling pathway in B lymphocytes. TGF–β–induced growth inhibition is the most extensively studied biological response to a TGF–β signal. Although in most cell types this response is mediated by Smad3– dependent regulation of c–Myc, p15<super>Ink4B</super>, and p21<super>Cip1</super> transcription, studies from Smad3 null mice suggest that TGF–β–induced growth inhibition in B lymphocytes occurs regardless of Smad3 status. We prove that this response does indeed occur independently of Smad3 in purified primary B lymphocytes and WEHI–231 cells. Consistent with this, p15<super>Ink4B</super> and p21<super>Cip1</super> are not noticeably induced by TGF–β in these cells, whereas Id3 and cyclin G2 are induced in a Smad3–independent manner. Finally, unlike the MAPK pathways we tested, the BMP–specific Smads 1 and 5 are activated in response to TGF–β in these cells, and this activation is dependent on ALK5 kinase activity. Collectively, these data indicate that TGF–β induces growth inhibition in B lymphocytes through a novel signaling pathway, and Smads 1 and 5 may help mediate this response.</p> / Dissertation

Keloids are a fibroproliferative disorder of unknown etiology developing in the skin after injury or spontaneously. The aim of this thesis is to gain deeper insight into the role of TGF-β and its signaling pathway proteins, SMADs, in the pathogenesis of keloids and describe the gene expression profile in different keloid sites in the search for potential target genes for future treatment. Further aim is to develop an instrument to describe the quality of life of patients with keloids. We find cultured keloid fibroblasts to express an increased level of TGF-β1 mRNA and a decreased level of TGF-β3 mRNA compared to control skin. Keloid derived fibroblasts exhibit significantly decreased mRNA levels of TGF-β receptor type II (TβRII) and the ratio of TβRI/TβRII mRNA expression is increased. This suggests that a certain expression pattern of TGF-β subtypes and receptors may be important in keloid pathogenesis. Analysis of keloid derived fibroblasts reveal decreased SMAD3 mRNA expression and decreased ratio of SMAD2/SMAD3 mRNA implicating a disturbed SMAD signaling. Keloid fibroblasts up-regulate SMAD4 protein after stimulation with TGF-β1 and display diminished levels of the inhibitory proteins SMAD6 and 7. This may contribute to unlimited and deregulated TGF-β signaling leading to increased extracellular matrix production (ECM). The gene expression pattern is described in fibroblasts from different keloid sites using microarrays covering the whole human genome. This study reveals 105 regulated genes (79 genes are up- and 26 down-regulated) resulting in a unique gene expression profile in different sites of keloids, where progression or regression of the keloid process took place. In cells from the central part of keloids with clinical signs of regression, an up-regulation of apoptosis inducing genes as ADAM12 and ECM degrading genes as MMP19 is found. These genes may contribute to regression of keloids and might be possible future target genes for treatment. Overexpression of apoptosis inhibitors as AVEN and down-regulation of angiogenesis inhibiting genes as PTX3 found at the active margin of keloids may be responsible for the invasive character of the keloid margin. We develop a disease specific questionnaire to measure the quality of life of patients with keloids. We find two scales, psychological and physical impairment, describing the dimensions of quality of life in patients with scars. These two scales are independent of each other and show a high test-retest reliability. Single items which clinically characterize the disease show correlations to these scales. The results of this study demonstrate for the first time a severe impairment of quality of life of patients suffering from keloids and hypertrophic scars. In conclusion the described alteration in TGF-β expression and its receptors, the disrupted SMAD signaling pathway and the unique gene expression patterns in different keloid sites provide new knowledge on ECM formation and degradation in keloids. Regulatory genes in ECM homeostasis may be future target genes for keloid prevention, regression and treatment. The disease specific quality of life instrument of patients with keloids and scars is a useful tool to estimate success in future therapeutic efforts over time.