Heparan sulfate proteoglycans modulate many of the growth factor pathways that drive prostate cancer progression. Prior to being secreted into the extracellular matrix, the covalently attached HS chains are modified by sulfation which has been shown to increase the affinity of binding growth factors. The specific HSPG that I focus on in this dissertation is Perlecan (Pln). Previously, our group along with collaborators found that 54 percent of prostate cancer tumors had upregulated levels of Pln protein that correlated with increasing Gleason score [93]. The LNCaP-DU145-LN4 cell line series is introduced as a model for this subset of tumors because Pln levels increase 50-fold as the cells become more metastatic. It was found that three stress-induced transcription factors, HIF1α, NFkB, and ATF2, all stimulate Pln expression. ChIP analysis reveals that HIF1α and NFkB directly bind the Pln promoter while ATF2 does not. The ROS-generating NADPH Oxidase and the ROS-inducible p38 MAPK were also found to induce Pln expression. To address the subset of prostate cancer tumors that reach metastasis without upregulation of Pln, I focused on the 2-o-sulfotransferase enzyme and its effect on proliferation and invasion in the LNCaP-C4-2B cell model which does not show upregulation of Perlecan expression. 2OST RNAi resulted in a significant decrease in proliferation in each line of the series. 2OST RNAi in highly metastatic C4-2B cells caused a significant decrease in cell invasion. Cells with decreased levels of 2OST had increased accumulation of actin and E-cadherin suggesting the possible formation of adherens junctions. I also found that expression of 2OST increases four-fold as cells become more metastatic. I found HIF1α and ATF2 act in a direct manner while NFkB acts indirectly to stimulate 2OST expression. In summary, I have analyzed the effect of cellular stress on the expression of the Pln and 2OST genes and investigated the phenotype of 2OST knockdown in metastatic prostate cancer cells. These studies lead me to propose that the tumor stress response is necessary for prostate cancer progression due to the role of stress in the upregulation of extracellular HS that is required for growth factor signaling and metastatic behaviors.

Developmental signaling pathways have many diverse roles throughout the life of an organism. The proper regulation of these pathways is essential for normal development, and misregulation can lead to diseases such as cancer. Heparan sulfate proteoglycans function to modulate growth factor signaling in many biological processes by acting as co-receptors, or by influencing ligand distribution. The heparan sulfate proteoglycan Trol, the Drosophila Perlecan homolog, is known to modulate signaling in a population of neuroblasts in the developing Drosophila central nervous system. My studies aim to determine the function Trol has in regulating signaling pathways during development. trol mutants are examined to determine how various mutant alleles impact signaling in several different developmental contexts. The role growth factor pathways play during induction of a Drosophila prostate cancer model is also examined. Gene expression profiles are determined for two types of prostate model overproliferation. Trol is shown to be able to differentially regulate multiple signaling pathways during several developmental processes. The Drosophila prostate cancer model is also shown to have many characteristics similar to those of human prostate cancer, and that signaling and proteoglycan expression are impacted by aberrant overgrowth in the model. My results indicate that Trol is able to specifically modulate different signaling pathways depending on the tissue and developmental context.

Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.

Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.

No description available.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.

Heparan sulfate proteoglycan (HSPG) or heparan sulfate (HS) degradation may contribute to endothelial cell (EC) dysfunction in diabetes. HSPGs, syndecan and perlecan, contain a protein core with mainly HS glycosaminoglycans (GAGs) attached. HSPGs modulate growth factors and function in membrane filtering. Heparanase induction is likely responsible for diabetic HS degradation. Heparin protects endothelium and insulin regulates glucose metabolism. Our objectives were to observe HSPG changes by studying EC GAG content and gene expression of syndecan, perlecan and heparanase under hyperglycemic conditions with insulin and/or heparin treatment.<p> GAGs, including HS, were determined by the carbazole assay and visualized by agarose gel electrophoresis in porcine aortic EC cultures treated with high glucose (30 mM) and/or insulin (0.01 U/ml) for 24, 48 and 72 hours and/or heparin (0.5 µg/ml) for 72 hours. High glucose decreased cell GAGs and increased medium GAGs. GAGs increased with time in control cultures and in high glucose plus insulin treated medium. GAGs were decreased with insulin but increased with insulin or heparin plus high glucose.<p> Confluent cultured human aortic ECs were incubated with control medium, high glucose and/or insulin and/or heparin for 24 hours. Real time PCR determination showed that: high glucose increased heparanase, decreased syndecan and had no effect on perlecan mRNA; insulin or heparin with/without high glucose decreased and insulin and heparin with high glucose increased heparanase mRNA; heparin and insulin with high glucose increased but insulin decreased syndecan mRNA. Actinomycin D (10 µg/ml) inhibited heparanase and syndecan mRNA with high glucose plus insulin plus heparin and inhibited heparanase mRNA with high glucose compared to time 0 but not â-actin after addition for 0, 2, 4, 8 and 24 hours. Bioinformatic studies revealed that transcription factor Sp1 activates heparanase promoter by high glucose and may play a role in regulation of perlecan and syndecan promoters.<p> Insulin or heparin inhibited the reduction in EC GAGs and syndecan mRNA and induction in heparanase by high glucose, indicating their protective effect. Decreased GAGs by insulin may relate to the pathology of hyperinsulinemia. Transcriptional regulation by heparin and/or insulin may cause variation in gene expression of heparanase, syndecan and perlecan.

Sperm protein, enterokinase and agrin (SEA) modules are small folds within large heavily glycosylated modular proteins. Because decreased expression of SEA-containing proteins such as perlecan (PLN) can lead to diseases such as Schwartz-Jampel syndrome (SJS), characteristics of the PLN SEA module including folding, potential for autocleavage, and protein binding were studied. Sequence analyses, recombinant protein evaluation, and a yeast two-hybrid screen were used to study the PLN SEA module and compare it to the mucin (MUC) 1 SEA module. In silico modeling of the PLN SEA module demonstrated a well conserved α/β sandwich fold. Experiments with expressed proteins showed that unlike MUC1, the PLN SEA module does not autocleave. Two-hybrid screening identified four “high confidence” proteins as potential binding partners which were explored in preliminary experiments. Together, these results demonstrate that PLN SEA module is unique and its properties cannot be generalized with other SEA module proteins such as MUC1.

Heparan sulfate proteoglycan (HSPG) or heparan sulfate (HS) degradation may contribute to endothelial cell (EC) dysfunction in diabetes. HSPGs, syndecan and perlecan, contain a protein core with mainly HS glycosaminoglycans (GAGs) attached. HSPGs modulate growth factors and function in membrane filtering. Heparanase induction is likely responsible for diabetic HS degradation. Heparin protects endothelium and insulin regulates glucose metabolism. Our objectives were to observe HSPG changes by studying EC GAG content and gene expression of syndecan, perlecan and heparanase under hyperglycemic conditions with insulin and/or heparin treatment.<p> GAGs, including HS, were determined by the carbazole assay and visualized by agarose gel electrophoresis in porcine aortic EC cultures treated with high glucose (30 mM) and/or insulin (0.01 U/ml) for 24, 48 and 72 hours and/or heparin (0.5 µg/ml) for 72 hours. High glucose decreased cell GAGs and increased medium GAGs. GAGs increased with time in control cultures and in high glucose plus insulin treated medium. GAGs were decreased with insulin but increased with insulin or heparin plus high glucose.<p> Confluent cultured human aortic ECs were incubated with control medium, high glucose and/or insulin and/or heparin for 24 hours. Real time PCR determination showed that: high glucose increased heparanase, decreased syndecan and had no effect on perlecan mRNA; insulin or heparin with/without high glucose decreased and insulin and heparin with high glucose increased heparanase mRNA; heparin and insulin with high glucose increased but insulin decreased syndecan mRNA. Actinomycin D (10 µg/ml) inhibited heparanase and syndecan mRNA with high glucose plus insulin plus heparin and inhibited heparanase mRNA with high glucose compared to time 0 but not â-actin after addition for 0, 2, 4, 8 and 24 hours. Bioinformatic studies revealed that transcription factor Sp1 activates heparanase promoter by high glucose and may play a role in regulation of perlecan and syndecan promoters.<p> Insulin or heparin inhibited the reduction in EC GAGs and syndecan mRNA and induction in heparanase by high glucose, indicating their protective effect. Decreased GAGs by insulin may relate to the pathology of hyperinsulinemia. Transcriptional regulation by heparin and/or insulin may cause variation in gene expression of heparanase, syndecan and perlecan.