Fibrosis is a disease where the normally transitory wound healing response enters a chronic state. Bleomycin and Adenovector models of pulmonary fibrosis have implicated TGF-β1 in this disease. Concern regarding a synergistic combination of TGF-β1 with an adaptive immune response within the Adenovector model prompted its use within mice devoid of T Lymphocytes, Balb/c SCIDs. The lack of an adaptive immune response within these mice did not affect the severity of fibrogenesis, as compared to Balb/c data in a hydroxyproline assay. TGF-β1 is a pluripotent cytokine with key roles in wound healing, immune regulation, and development, making it a dangerous molecule to therapeutically modulate directly. Future strategies will likely focus on downstream fibrotic molecules uninvolved in immune regulation, such as CTGF. While CTGF has been associated with fibrosis and is likely activated by TGF-β1, no conclusive evidence is available within an animal model. TGF-β1 stimulates cells by binding its receptor and signaling through the Smad signal transduction pathway. Smad3 knockout mice were used to examine the regulation of CTGF by TGF-β1, and study its role in pulmonary fibrosis. We show that these mice produce dramatically less CTGF in response to TGF-β1 than littermates expressing Smad3, and they show protection against TGF-β1 induced pulmonary fibrosis, using the Adenovector system. TGF-β1 can alter lung development, and is thought to be a causative agent in Bronchopulmonary Dysplasia, a disease affecting immature lungs. Utilizing the Adenovector system, we developed a neonatal rat model of BPD that closely resembles the human disease, providing researchers with a system to study the disease course. TGF-β1 is part of a family of growth factors, of which TGF-β3 is also a member. What role TGF-β3 plays in pulmonary fibrosis has not been evaluated. To allow future in vivo studies on the effect of TGF-β3 on lung morphology, we constructed a replication deficient Adenovector expressing constitutively active TGF-β3. / Thesis / Master of Science (MSc)

No description available.

One of the most frequently reported deficits seen in individuals with Fetal Alcohol Spectrum Disorder (FASD) is impairments in learning and memory, which is likely attributed to the teratogenic effects of ethanol on the developing hippocampus. TGF-β (transforming growth factor-β), hedgehog and Wnt signaling pathways have been identified as high probability candidate pathways associated with brain deficits seen in FASD. Increasing evidence indicates that ethanol may induce changes in DNA methylation that could alter transcription regulating factors within signaling pathways critical in brain development. The purpose of this study was to test the hypotheses that prenatal ethanol exposure during i) the first trimester-equivalent period, or ii) throughout the entire gestational period induces changes in DNA methylation and alters the transcription/translation of TGF-β1, SHH (sonic hedgehog) and Wnt3a within the developing hippocampus. Pregnant Dunkin-Hartley-strain guinea pigs were assigned to one of three groups: ethanol (4 g/kg maternal body weight), isocaloric-sucrose/pair-feeding, or no treatment. Embryonic telencephalon tissue (which gives rise to the hippocampus) and fetal hippocampus were collected at gestational day (GD) 23 or GD 65, respectively. GD 23 ethanol-exposed and nutritional control embryos exhibited decreased crown-rump and head lengths. GD 65 ethanol-exposed fetuses exhibited decreased body and brain weights compared with the control groups. Ethanol exposure during the first trimester-equivalent period, but not during the entire gestational period, resulted in an increase in global DNA methylation. First trimester-equivalent ethanol exposure did not alter TGF-β1, SHH and Wnt3a gene expression within the GD 23 telencephalon. However, ethanol exposure throughout the entire pregnancy led to an increase in the expression of all three genes within the GD 65 hippocampus. No change in TGF-β1 protein was seen in the hippocampus of ethanol-treated fetuses. Post-translationally modified (ptm) SHH, but not unmodified SHH protein, was decreased in the hippocampus of ethanol-exposed fetuses. A decrease in unmodified, but not ptm Wnt3a protein, was observed in both ethanol-exposed and nutritional control hippocampus. These results suggest that prenatal ethanol exposure may affect hippocampal development through alterations in i) DNA methylation as shown at early gestation and ii) the expression of transcription regulating factors, especially SHH, as shown at term. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-12-03 12:36:33.035

Inactivation of p53 by mutation occurs in half of human tumours. The majority of these mutations affect the DNA-binding core domain and hence impair DNA binding and hinder transcription of p53 target genes. A wealth of data indicates that even cancers carrying wild type p53 protein, evolve mechanisms to render the p53 pathway inactive. Thus, inactivation of the p53 response, either by mutation or the alternative mechanisms, allows unpurturbed tumour growth. Recent work identified Anterior Gradient-2 (AGR2) as a protein overexpressed in wild type p53 expressing tumours and it was subsequently shown that AGR2 inhibits p53 pathway. In this study I confirmed that AGR2 protein inhibits p53 and AGR2 depletion potentiates p53-dependent DNA damage response. As there were no physiological signals known that regulate the AGR2-p53 axis, here I set out to identify pathways that activate or inhibit AGR2. I found that transforming growth factor β(TGF- β) triggers AGR2 protein reduction and this is concomitant with the stabilisation and increased activity of p53 protein. TGF-β halts AGR2 transcription in a SMAD4- dependent manner and triggers AGR2 protein degradation involving an ATM kinase. I found that SMAD nuclear interacting protein (SNIP1) mediated the ATMdependent AGR2 degradation. Interestingly, SNIP1 overexpression by itself promoted AGR2 protein degradation. I found that AGR2 protein degradation was proteasome independent and involed autophagy-lysosomal degradation pathway. As the mechanism of p53 inhibition by AGR2 is not known, I reasoned that identifying interactors of AGR2 may potentially further our understanding of the mechanism accounting for AGR2-mediated p53 inhibtion. I isolated the ATP binding protein Reptin in the yeast two-hybrid system and subsequently validated it as an AGR2 binding partner. Mutations of the two ATP binding motifs in Reptin resulted in altered oligomerization and thermostability of Reptin and affected the AGR2-Reptin complex stability. I also identified the Reptin docking site and it was mapped to a divergent octapeptide loop. I found that AGR2-Reptin complex coimmunoprecipitated with the p53 protein. Subsequently, I showed that Reptin protein can influence p53 activity, and depending on local concentration, either inhibit the transcription of p53-genes or chaperone its DNA binding activity. Interestingly, I found that Reptin formed a stable complex, independent of AGR2, with p53 R175H, p53 F270A, p53 S269D and p53 S269A, which has implication for the Reptin function in cancers bearing mutant form of p53 protein.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

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

Intestinal immune cells remain tolerant to the trillions of commensal bacteria present in the gut, with perturbations of this process implicated in development of inflammatory bowel disease (IBD). The cytokine TGF-β is a key factor promoting intestinal immune tolerance, but is secreted in a latent state that requires activation to function. Binding of TGF-β to the integrin αvβ8 is a principal mechanism of TGF-β activation, with mouse models demonstrating a crucial role for αvβ8 expression by dendritic cells and regulatory T cells in intestinal immune regulation. Despite this evidence, very little is known regarding the importance of this activating integrin in human intestinal homeostasis. Utilising flow cytometry here we find that integrin αvβ8 is highly expressed on peripheral blood monocytes with highest levels on intermediate CD14++CD16+ monocytes. Upon monocyte to macrophage differentiation high β8 expression is observed on anti-inflammatory M-CSF differentiated macrophages versus pro-inflammatory GM-CSF macrophages. In monocytes, expression of β8 is upregulated by specific bacterial TLR ligands. Utilising a TGF-β reporter cell line both monocytes and M-CSF MDM display an enhanced ability to activate TGF-β in an αvβ8-dependent manner. Data presented here indicate that macrophage αvβ8-dependent TGF-β activation does not alter expression of surface markers associated with a tolerogenic macrophage phenotype, phagocytosis, or production of the anti-inflammatory cytokine IL-10; nor does TGF-β appear to influence the metabolic profile of macrophages, key differences of which are associated with pro- or anti-inflammatory phenotype. However, the previously undescribed finding of integrin αvβ8 expression on human monocytes and macrophages, which was subsequently confirmed in intestinal populations and found to be downregulated in inflamed IBD mucosa, may highlight an important functional pathway in intestinal immune homeostasis and represent a potential future therapeutic target in IBD.

Endometriosis is a benign inflammatory disorder, defined by the presence of endometrial tissue outside the uterus with lesions typically found on the pelvic peritoneum in close association with the peritoneal mesothelium. The prevalence of endometriosis is estimated at 6-10% of women of reproductive age and it is associated with chronic pelvic pain, dysmenorrhoea, dyspareunia and infertility. Surgical excision can provide symptom relief, but symptoms recur in up to 75% of surgical cases and available medical treatments have undesirable side effects. New treatments are limited due to our poor understanding of the aetiology of endometriosis. To date, the majority of research has focused on changes within the ectopic endometrial tissue to explain the development of endometriosis lesions, however, there is increasing evidence that the peritoneal mesothelium plays an important role. According to Sampson’s theory of retrograde menstruation, ectopic endometrial cells must first have to attach to the surface of the peritoneum before undergoing invasion, proliferation, and neoangiogenesis. TGF-β1 is an inflammatory growth factor that regulates a variety of cellular functions including; cell adhesion, cell invasion and angiogenesis. Levels of TGF-β1 are increased in the peritoneal fluid of women with endometriosis compared to controls and research using a mouse model of endometriosis has demonstrated TGF-β1-null mice to develop smaller and fewer endometriosis lesions than their wild-type controls. Together these studies suggest that TGF-β1 plays a major role in the development of peritoneal endometriosis lesions and that targeting this pathway may be of therapeutic potential. However the functional role that TGF-β1 plays in peritoneal endometriosis is still unclear. The overall aim of this thesis was to determine if the peritoneal expression of TGF-β and its target genes are disrupted in women with endometriosis and whether this could contribute to the development of endometriosis lesions. Our first aim was to determine if the peritoneum was a source of TGF-β expression and if reception and/or signalling were altered in women with endometriosis. We found that the peritoneal fluid of women with endometriosis contained increased concentrations of TGF-β1 and that peritoneal mesothelial cells adjacent to endometriosis lesions expressed significantly higher levels of TGF-β1 mRNA. Analysis of TGF-β signalling targets within the peritoneum showed that women with endometriosis express significantly higher levels of TGF-β targeted genes associated with tumourigenesis processes including; EMT, invasion and angiogenesis. We next asked if there are changes in the metabolic phenotype of endometriosis lesions and peritoneum in women with endometriosis, similar to the metabolic changes seen in tumour cells. Endometriosis lesions expressed markers of aerobic glycolysis, including HIF-1α, suggesting that lesions may metabolise in a similar fashion to tumours. Furthermore, peritoneum adjacent to endometriosis lesions expressed significantly higher levels of markers of aerobic glycolysis, including HIF-1α, suggesting that the peritoneum may feed forward high-energy lactate, a by-product of glycolysis, to the endometriosis lesions. These observations were supported by significantly increased lactate concentrations within the peritoneal fluid of women with endometriosis that positively correlated with levels of TGF- β1. TGF-β1 was shown to increase expression of glycolysis markers and lactate expression in peritoneal mesothelial cells in-vitro, suggesting TGF-β may regulate this change. We then determined if TGF-β1 was responsible for the change in peritoneal mesothelial cell metabolism by signalling through the ID-HIF-1α pathway. ID proteins are transcription factors, whose expression is regulated by the TGF-β superfamily. We found expression of ID1 mRNA to be increased in the peritoneum of women with endometriosis and that TGF-β1 significantly increased ID1 but decreased ID2 expression in the peritoneal mesothelial cells in-vitro. ID1 siRNA knockdown decreased glycolysis initiator HIF-1α mRNA and ID2 siRNA knockdown increased HIF-1α mRNA and lactate expression, suggesting TGF-β1 regulates mesothelial cell metabolism, at least in part, through the ID pathway. ID transcription factors are also known to regulate VEGF-A expression, therefore we next determined if TGF-β1 induced ID1 and/or reduced ID2 expression in the peritoneum promoted VEGF-A mRNA and protein expression. VEGF-A, a cytokine essential for angiogenesis, was significantly increased in the peritoneal fluid of women with endometriosis and levels positively correlated with TGF-β1. TGF-β1 increased VEGF-A expression in-vitro and siRNA knockdown of ID1 decreased and siRNA knockdown of ID2 increased VEGF-A mRNA and protein expression in the peritoneal mesothelial cell. Lastly we aimed to determine if TGF-β1 induces EMT in peritoneal mesothelial cells. The peritoneum of women with endometriosis expressed higher levels of EMT markers. Exposure of peritoneal mesothelial cells to TGF-β1 in-vitro induced EMT-like changes, including; changes to cell morphology, gene expression and protein localisation. Peritoneal mesothelial cells were more migratory and invasive suggesting that TGF-β1 induced EMT may disrupt the mesothelial cell monolayer allowing ectopic endometrial cells to invade the peritoneal tissue or for peritoneal mesothelial cells to migrate into endometriosis lesions. In summary, the novel data presented in this thesis provides evidence that the pelvic peritoneum and in particular the peritoneal mesothelial cell may play a critical role in the aetiology of peritoneal endometriosis. Expression of TGF-β1 and its transcriptional target genes are dysregulated in the peritoneum of women with endometriosis. TGF-β regulated ID expression may induce changes in cell metabolism and promote neoangiogenesis, prompting peritoneal endometriosis lesion development. Furthermore other TGF-β1 transcriptional targets, such as those involved in EMT, are also altered in the peritoneum of women with endometriosis and may contribute the development and maintenance of lesion formation. These results point to a central role for TGF-β1 expression and signalling in the aetiology of peritoneal endometriosis. Furthermore it is likely that changes within the expression profile and morphology of the peritoneal mesothelial cells contribute to peritoneal lesion formation. Drugs that target these pathways may provide new therapies for women with endometriosis.

The transforming growth factor-ß (TGFß) pathway, including the bone morphogenetic protein (BMP), plays critical roles during embryogenesis and in adult tissue homeostasis. Hence, malfunctions in TGFß/BMP signalling result in several diseases. Signalling is initiated by ligand binding to cell surface receptor kinases, which phosphorylate and activate the R-SMAD transcription factors. R-SMADs translocate to the nucleus and regulate the transcription of hundreds of genes. The cellular responses to TGFß/BMP signals are tightly controlled and highly regulated. TGFß/BMP receptors and R-SMADs, as the intracellular mediators of TGFß/BMP ligands, are key targets for regulation to control duration and potency of signalling. Reversible ubiquitylation of R-SMADs and TGFß/BMP receptors is a key mechanism to control TGFß/BMP signalling. Several E3 ubiquitin ligases have been reported to regulate the turnover and activity of TGFß/BMP receptors and R-SMADs, however little is known about their cognate deubiquitylating enzymes (DUBs). A proteomic screen identified the DUBs OTUB1 and USP15 as potential novel regulators of the TGFß and BMP pathways respectively. Endogenous OTUB1 was recruited to the active phospho-SMAD2/3 complex only upon TGFß induction and OTUB1 had a crucial role in TGFß-mediated gene transcription and cellular migration. OTUB1 inhibited the ubiquitylation of phospho-SMAD2/3 by binding to and inhibiting the E2 ubiquitin-conjugating enzymes independently of its catalytic activity. Consequently, the depletion of OTUB1 in cells caused a rapid loss in levels of TGFß-induced phospho-SMAD2/3, which was rescued by the proteasomal inhibitor Bortezomib. These findings demonstrated a novel signal-induced phosphorylation-dependent recruitment of OTUB1 to its target. Hence, OTUB1 could be exploited as a target to intervene against diseases that are provoked by an imbalance in TGFß signalling. DUBs are highly regulated enzymes and recent reports have shed light into the molecular regulation OTUB1. The N-terminal region of OTUB1 harbours an ubiquitin binding domain, which is critical for its function to inhibit ubiquitylation. While investigating the role of OTUB1 in TGFß signalling, it became apparent that OTUB1 itself could be post-translationally modified by phosphorylation. Two phosphorylation sites at the OTUB1 N-terminal region have been identified by mass spectrometry. S18 of OTUB1 was phosphorylated in vitro by the type I TGFß receptor (ALK5), whereas S16 was phosphorylated by the constitutively active kinase CK2 in vitro and in vivo. Phosphorylation of the OTUB1 N-terminal region could affect its physiological function and requires further investigation. Although much is known about DUBs that target the type I TGFß receptor, no DUBs that target the type I BMP receptors had been identified. USP15 was identified in a proteomic screen as an interactor of SMAD6, which is a negative regulator of the BMP pathway. USP15 also binds to and deubiquitylates the type I BMP receptor (ALK3), thereby enhancing BMP signalling. Consequently, USP15 impacts BMP-induced SMAD1 phosphorylation, mouse osteoblastic differentiation and Xenopus embryogenesis. A proteomic approach identified O-GlcNAc transferase (OGT) as an interactor of SMAD2. SMADs have not been associated with O-GlcNAc modifications and the regulation of TGFß/BMP signalling by O-GlcNAcylation has not been investigated. Endogenous SMADs1-3 bound OGT and pulled down potential O-GlcNAc modified proteins. Furthermore, SMAD4 was possibly O-GlcNAcylated, which implies that O-GlcNAc modification could regulate TGFß/BMP signalling. Further investigation is needed to decipher the precise molecular mechanisms of this potential regulation.

Background: Different types of cardiomyopathies are associated with variable hypertrophic response. A number of growth factors are thought to play a role in pathologic cardiac remodeling. Aims: We compared the modulation of the TGF-ƓÒ superfamily and IGF-1 signaling pathways and their target genes, the cell cycle regulatory proteins in tachycardia-induced dilated cardiomyopathy, a model with no detectable hypertrophy and in ischemic cardiomyopathy, a model with a marked hypertrophic reaction. Methods: In the first study, endomyocardial biopsies were obtained weekly in 15 dogs, during the development of tachycardiomyopaty. Genes involved in the myostatin-TGF-ƓÒ-Activin-A/Smad signaling pathway, p21 and cyclin D were quantified and correlated to echocardiographic measures of hypertrophy. In the second study, myocardial tissue samples were obtained in 8 dogs with a healed myocardial infarction, in 8 dogs with heart failure induced by overpacing and in 7 healthy dogs. We measured gene expression of IGF-1, its receptor (IGF-1R) and cyclins A, B, D1, D2, D3 and E and correlated them to the level of hypertrophy. Results: Tachycardiomyopathy was characterized by chambers dilation with no identifiable hypertrophy. Ischemic cardiomyopathy was characterized by eccentric hypertrophy. In tachycardiomyopathy, Activin-A mRNA was 4-fold higher than at baseline. Smad7 was overexpressed in severe heart failure; p21, a direct target gene of the Smad pathway was upregulated 8-fold and cyclin D1 was down-regulated. In that model, IGF-1 was overexpressed but neither IGF-1R nor any of the cyclins studied. In ischemic cardiomyopathy, IGF-1, IGF-R, and cyclins B, D1, D3 and E gene expression were upregulated. In tachycardiomyopathy, Activin-A and p21 were inversely correlated to the thickness of the interventricular septum. In normal dogs and in the both models of cardiomyopathy, IGF-1R was correlated to the thickness of the interventricular septum and to cyclins. Conclusions: Taken together, these results agree with the notion that Activin-A, IGF and cyclins are involved in the modulation of hypertrophic response observed in cardiomyopathies.