Androgen receptor (AR) is a member of the steroid hormone receptor family of molecules, and expansion of a CAG repeat encoding polyglutamine (poly-Q) in AR gene are associated with a progressive neuromuscular disease known as spinal bulbar muscular atrophy (SBMA) or Kennedy disease. The hallmark of SBMA diseases is formation of juxtanuclear AR inclusions that have been termed ¡¥AR aggregates¡¦.Previous studies showed that transgenic mice overexpressing wild-type AR exclusively in the skeletal muscle fibers display similar abnormalities to those observed in models of SBMA disease. To elucidate the mechanisms underlying toxicity conferred by wild-type protein aggregation within normal cells, a mouse embryonic stem cell (ESC) model with non-genetic modified settings in AR overexpression was used to display the common features of polyglutamine disease in this experiment. It was found that wild-type AR proteins are highly expressed and form nuclear aggregate inclusions in response to androgen treatment in ES cells, the formation of AR aggregates inhibit the differentiation of embryonic bodys and enhanced caspase-3 activity in androgens -induced apoptosis. In addition, it was also investigated that relation between chaperones¡BAR and the endoplasmic reticulum (ER) stress-induced pathways in ES cells in this study, and it was found that chaperones could colocalize with AR aggregates, these findings may help us to better understand the roles of the chaperones on AR aggregates.

The unfolded protein response (UPR) is a well conserved mechanism in eukaryotes that protects organisms from the damaging effects of endoplasmic reticulum (ER) stresses. Activation of the UPR will lead to two outcomes. It first attempts to restore cellular functions by enhancing protein folding capacity, inhibiting protein synthesis and promoting degradation of harmful proteins (the pro-survival pathway). However, if the stressful conditions are prolonged or severe, apoptosis will be induced (the pro-apoptotic pathway). The present study suggests that an isoform of the cellular protein Zhangfei (ZF, CREBZF) is linked to the pro-apoptotic pathway in the UPR by using DNA, protein and cell viability analyses. This isoform is known as the short-tail ZF (stZF). We demonstrated that stZF can be induced by prolonged ER stress. The protein of stZF is stable under ER stress and it has the ability to promote programmed cell death in the early stage of apoptosis through the induction of CHOP, a protein that plays a key role in the pro-apoptotic pathway of the UPR.

Beukes Hip Dysplasia (BHD) is an autosomal dominant disorder where the abnormal shape of the hip joint leads to secondary osteoarthritis. The locus of BHD has been previously mapped to 4q35 and screening of candidate genes within this region revealed a mutation in the gene encoding the Ubiquitin-fold modifier 1 specific protease 2 (Ufsp2). The mutation prevents Ufsp2 from cleaving its target, Ufm1. Ufsp2 and Ufm1 are both components of a novel ubiquitin-like protein modification system which involves Ufm1 being processed via the E1, E2 and E3 enzymes (Uba5, Ufc1 and Ufl1, respectively) and attachment to target protein(s) one of which has been identified (Ddrgk1). The aim of this study was to investigate the link between the UFSP2 mutation and the BHD phenotype by: (i) examining the expression of components of the Ufm1 system in vivo and in vitro; (ii) development of an in vitro Ufm1 conjugation system; and (iii) generation and analysis of transgenic mice overexpressing the Ufsp2 gene with the BHD mutation. The expression of Ufsp2 was determined by radioactive RNA in situ hybridisation of mouse tissue sections. The analysis revealed Ufsp2 expression predominantly in the bone of the hip joint and in the bone and secondary ossification centres of the knee of 10 day old mice. Real-Time PCR analysis showed increased expression of components of the Ufm1 system during in vitro osteogenic and chondrogenic differentiation which coincided with induction of ER stress evidenced by upregulation of Bip. These components were also upregulated in response to chemically induced ER stress in vitro. Analysis of the promoter regions of Ufm1 system genes identified unfolded protein response elements in the upstream sequences of Uba5, Ufl1, Ufm1 and Lzap genes and the elements in Uba5 and Lzap were found to be required and responsive to ER stress using luciferase promoter assays. A Tandem Affinity Purification method was developed for isolation and identification of Ufm1 conjugation targets from cell lines expressing modified forms of Ufm1. Mass spectrometry analysis of Ufm1 conjugates purified from HEK293T cell line identified Uba5 and Ufc1 but no new Ufm1 targets. Western blot comparison of Ufm1 conjugated proteins purified from the HEK293T and 2T3 osteoblast cell lines identified putatative Ufm1 conjugation targets and increased conjugation in osteoblasts in response to ER stress. One of these targets was identified as Ddrgk1 but the remaining putative targets remain to be identified by mass spectrometry. Transgenic mice overexpressing the mutated Ufsp2 gene were generated and subjected to phenotypic analysis. No significant differences were found between transgenic and wild type mice following X-ray, histological and weight analysis. Higher expression of Ufsp2 in bone and secondary ossification centres as well as upregulation of components of the Ufm1 system in response to ER stress suggests that the molecular pathway between the UFSP2 mutation and the BHD phenotype may relate to abnormal ER stress responses during osteoblast differentiation. Further studies are however required to determine how the Ufm1 system modulates ER stress responses and how disruption of these processes caused by the UFSP2 mutation causes BHD.

Thymic stromal lymphopoietin (TSLP) produced by epithelial cells acts on dendritic cells (DCs) to drive differentiation of TH2-cells, and is therefore important in allergic disease pathogenesis. However, DCs themselves make significant amounts of TSLP in response to microbial products, but little is known about the key downstream signals that induce and modulate this TSLP secretion from human DCs. We show that human monocyte derived DC (mDC) secretion of TSLP in response to Candida albicans and β-glucans requires dectin-1, Syk, NF-κB, and p38 MAPK signaling. In addition, TSLP production by mDCs is greatly enhanced by IL-1β, but not TNF-α, in contrast to epithelial cells. Furthermore, TSLP secretion is significantly increased by signals emanating from the endoplasmic reticulum (ER) stress response, specifically the unfolded protein response sensors, inositol-requiring transmembrane kinase/endonuclease 1 and protein kinase R-like ER kinase, which are activated by dectin-1 stimulation. Thus, TSLP production by mDCs requires the integration of signals from dectin-1, the IL-1 receptor, and ER stress signaling pathways. Autocrine TSLP production is likely to play a role in mDC-controlled immune responses at sites removed from epithelial cell production of the cytokine, such as lymphoid tissue.

The number of people living with diabetes worldwide is continually increasing. The majority of these people will eventually die of cardiovascular disease, the major underlying cause of which is atherosclerosis. Despite the efforts of many researchers, gaps in our knowledge still exist regarding the molecular mechanism(s) linking the two conditions. Current data suggests that the hexosamine biosynthetic pathway (HBP) may have a role in the development of hyperglycemia-accelerated atherosclerosis. About 2-3% of glucose entering a cell is diverted into this pathway where it is modified through a series of reactions to yield the end product, UDP-N-acetylglucosamine (UDP-GlcNAc); a substrate for both N- and O-linked glycosylation of various molecules. N-linked glycosylation occurs in the endoplasmic reticulum (ER) and is an important process in the maintenance of ER homeostasis. We hypothesized that a dysregulation in the HBP can ultimately trigger ER stress – an event associated with the development of atherosclerosis. We have established a method that allows us to monitor levels of UDP-GlcNAc both in cultured cells and mouse tissues through high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Using this technique, we’ve shown that both glucosamine supplementation and overexpression of the rate limiting enzyme of the HBP, GFAT, in cultured cells results in elevated UDP-GlcNAc levels. Furthermore, glucosamine was shown to trigger ER stress. In contrast, three GFAT inhibitors that were previously identified in a high throughput screen were shown to decrease UDP-GlcNAc levels and one inhibitor, dehydroiso-β-lapachone, appears to prevent ER stress induction. Finally, we use complementary methods to show that the HBP is augmented in the livers of hyperglycemic mice. This process may play a role in the accelerated development of atherosclerosis. Together, these results provide further insight into the role of the HBP in diabetic atherosclerosis and the established methods provide a platform for the further investigation of this mechanism. / Thesis / Master of Science (MSc)

Major diseases such as cardiovascular diseases, diabetes, obesity and tumor growth are known to involve inflammatory angiogenesis. MCP-induced protein 1 (MCPIP1) encoded by ZC3H12A gene, was reported to promote angiogenesis and is addressed in my dissertation as MCPIP. The mechanism/s involved in the angiogenic differentiation induced by MCPIP was however unknown. The aim of this study was to bridge this gap in our knowledge and delineate the molecular mechanisms and sequential processes involved in angiogenesis mediated via MCPIP. To determine if angiogenesis induced by inflammatory cytokines, TNF-, IL-1 and IL-8 is mediated via induction of MCPIP, knockdown of MCPIP by its specific siRNA, in human umbilical vein endothelial cells was performed. Oxidative stress, ER stress and autophagy are known to be involved in mediating inflammation. We hypothesized that MCPIP-induced angiogenic differentiation is mediated via induction of oxidative stress, ER stress and autophagy. Chemical inhibitors and specific gene knockdown approach were used to inhibit each process postulated. Oxidative stress was inhibited by apocynin or cerium oxide nanoparticles or knockdown of NADPH oxidase subunit, phox47. Endoplasmic reticulum (ER) stress was blocked by tauroursodeoxycholate or knockdown of ER stress signaling protein IRE-1 and autophagy was inhibited by the use of 3methyl adenine, or LY 294002 or by specific knockdown of beclin1. Matrigel assay was used as an in vitro tool to assay angiogenic differentiation. Inhibition of each step inhibited the subsequent steps postulated. The results reveal that angiogenesis induced by inflammatory agents is mediated via sequential induction of MCPIP that causes v oxidative and nitrosative stress resulting in ER stress leading to autophagy required for angiogenesis. MCPIP has deubiquitinase and anti-dicer RNase activities. If and how the dual enzymatic activities of MCPIP mediate angiogenesis was unknown. Our results showed that hypoxia-induced angiogenesis is mediated via MCPIP. MCPIP deubiquitinated ubiquitinated hypoxia-inducible factor (HIF-1) and the stabilized HIF-1 entered the nucleus to promote the transcription of its target genes, cyclooxygenase-2 and vascular endothelial growth factor causing the activation of p38 MAP kinase involved in angiogenesis. MCPIP expression promoted angiogenesis by inhibition of thrombospondin-1 synthesis via induction of silent information regulator (SIRT)-1 and/or via suppression of VEG-inhibitor levels caused by inhibition of NF-B activation. MCPIP inhibited the production of the anti-angiogenic microRNAs (miR)-20b and miR-34a that repress the translation of HIF-1 and SIRT-1, respectively. Cells expressing the RNasedead mutant of MCPIP, D141N, that had lost the ability to induce angiogenesis had deubiquitinase activity but did not inhibit the production of miR-20b and miR-34a. Mimetics of miR-20b and miR-34a inhibited MCPIP-induced angiogenesis. These results show for the first time that both deubiquitinase and anti-dicer RNase activities of MCPIP are involved in inflammatory angiogenesis. Results from our study delineate key processes that could be potential targets for therapeutic intervention against inflammatory angiogenesis.

Damage to the endothelium is an important contributor to the initiation and progression of atherosclerosis. GRP78 is an endoplasmic reticulum (ER)-resident molecular chaperone in normal healthy endothelium that functions to assist in the correct folding of newly synthesized proteins and to prevent the aggregation of folding intermediates. In addition, GRP78 is present as a transmembrane protein on the surface of lesion-resident endothelial cells. Surface GRP78 is known to act as a surface signaling receptor in cancer cells and is activated by anti-GRP78 autoantibodies (GRP78a-Abs) isolated from the serum of cancer patients. However, the role of cell surface GRP78 on endothelial cells and the influence of GRP78a-Abs in atherosclerosis is unknown. The objectives of this study were to investigate the effects of GRP78a-Abs on lesion development, examine whether engagement of cell surface GRP78 by GRP78a-Abs modulates endothelial cell function, and determine whether GRP78a-Abs were associated with cardiovascular disease (CVD) in humans. This research showed that ApoE-/- mice with advanced atherosclerotic lesions have elevated serum levels of GRP78a-Abs and ApoE-/- mice immunized against recombinant GRP78 demonstrated a significant increase in GRP78a-Abs titers as well as accelerated lesion growth. Furthermore, this work demonstrated that activation of surface GRP78 on endothelial cells by GRP78a-Abs significantly increases gene expression of adhesion molecules ICAM-1 and VCAM-1 as well as leukocyte adhesion through the NFκB pathway. Additionally, middle-aged to elderly adults at risk for CVD showed a tendency toward elevated circulating GRP78a-Ab levels. Our results suggest that signaling through cell surface GRP78 can activate intracellular pathways that contribute to endothelial cell activation and augment atherosclerotic lesion development. These findings demonstrate a novel role for GRP78a-Abs and surface GRP78 receptor activity in endothelial cell function and the early stages of lesion development, as well as establish an initial framework for future work involving circulating GRP78a-Abs and atherosclerotic disease in humans. Furthermore, this work indicates inhibiting the interaction of GRP78a-Abs with cell surface GRP78 could present a novel therapeutic strategy to modulate lesion growth, thereby reducing the risk for atherosclerosis and cardiovascular disease. / Thesis / Doctor of Philosophy (PhD)

No description available.

ER stress occurs in response to the accumulation of unfolded or misfolded proteins in the ER lumen, subsequently activating three signal transduction pathways collectively called the unfolded protein response (UPR). Although the goal of the UPR is to restore ER homeostasis, it can result in apoptosis when ER stress is too severe or prolonged. CHOP, which is induced by all three branches of the UPR, is central to inducing ER stress-induced apoptosis by regulating the expression of numerous pro-apoptotic genes. One of the downstream targets of CHOP is GADD34, which contributes to PP1-mediated dephosphorylation of eIF2α together with its homolog CReP to restore the attenuated global translation. A previous study reveals that morpholino knockdown of GADD34 rescued chronic ER stress-induced apoptosis in zebrafish caudal fin epidermal cells in vivo. However, due to the limitations of morpholino, any morpholino data about novel phenotypes should be verified with comparative mutant data. Therefore, we sought to investigate how GADD34 and CReP are involved in acute and chronic ER stress-induced apoptosis using GADD34 or CReP mutant zebrafish embryos. GADD34 heterozygous or CReP heterozygous zebrafish are crossed to produce the embryos of the following genotypes: wildtype, GADD34 heterozygous mutant, GADD34 homozygous mutant, CReP heterozygous mutant, and CReP homozygous mutant embryos. At 24hpf, embryos were treated with ER stress inducer Thapsigargin for either 4 hours or 24 hours to induce acute or chronic ER stress. After stained with acridine orange for an apoptosis assay, each embryo was imaged under a confocal microscope and subsequently genotyped. GADD34 or CReP mutation alone did not affect levels of apoptosis induced by acute ER stress. Similarly, CReP mutation alone did not affect levels of apoptosis induced by chronic ER stress. However, GADD34 mutation rescued chronic ER stress-induced apoptosis, and the differences between the apoptosis level in GADD34 homozygous mutants and those in wildtype and GADD34 heterozygous mutants were statistically significant. Our results indicate no obvious role of GADD34 and CReP in acute ER stress-induced apoptosis, which is consistent with the previous morpholino data. However, as GADD34 mutation rescued chronic ER stress-induced apoptosis, the CHOP- GADD34 downstream pathway is likely to be involved in chronic ER stress-induced apoptosis. In the future, GADD34 and CReP double mutant embryos should be used to analyze for the redundancy of GADD34 and CReP.

In this project I sought to analyse the effects of different free fatty acids (FFAs) on INS-1E β-cells. The saturated fatty acid palmitate is considered toxic whereas the monounsaturated fatty acid oleate is harmless. In my working hypothesis I assumed an additional protective effect of oleate when used in combination with palmitate. Furthermore I aimed to explore in detail the possible causes and signalling pathways responsible for apoptosis or sustained cell survival. I examined the Endoplasmic Reticulum (ER) stress response, called unfolded protein response (UPR), as one essential criterion deciding about cell death or life. Analysis of viability and apoptosis confirmed the deleterious effect of palmitate on INS-1E β-cells after 24h of incubation. Oleate proved not to be harmful and even reversed the toxicity of palmitate. When the main components of the UPR were assessed using Western blot analyses and quantitative PCR was performed I found positive proof that palmitate activated the UPR and ultimately led to apoptosis. By contrast, oleate completely prevented UPR signalling. I conclude that oleate rescues INS-1E β-cells by inhibiting ER stress and its signalling.