Conditions that lead to reversible growth arrest (quiescence), promote the expression of a set of genes called growth arrest specific (GAS) genes. GAS genes play a crucial role in initiating and maintaining the entry into quiescence, while also activating stress responses to help the cell overcome the effects of the stressors. Gene profiling study examining the transcriptome has shown a vast number of genes that are upregulated during quiescence, among them is GEM1 (GTP binding protein overexpressed in skeletal muscle). GEM1 transcripts were elevated 18-fold in response to quiescence. GEM1 is a small monomeric GTPase from the Ras superfamily. It is involved in regulation of cytoskeleton reorganization, and inhibition of voltage gated calcium channels that ultimately prevents hormone secretion. A preliminary study determined that GEM1 is packaged into extracellular vesicles (EV). GEM1 is also reported to promote lipid accumulation and adipogenesis in goat pre-adipocytes. GEM1 is also reported to bind transcription factors that are involved in lipid homeostasis pathways. Thus, it is probable that GEM1 may play a major role in EV formation and/or release, and lipid homeostasis. This study examined the expression of GEM1 at the protein level and validates its candidacy as a GAS gene. We also created two GEM1-shRNA retroviral constructs capable of partially downregulating GEM1 expression which can serve as a molecular tool for further characterizing the function of GEM1 in quiescent CEF. / Thesis / Bachelor of Science (BSc) / GEM1 is a small monomeric GTPase, implicated in a variety of roles in eukaryotes. It plays a role in regulating adipogenesis, and hormone secretion. Most notably it regulates cytoskeleton reorganization in response to changes in calcium concentrations. Gene profiling done by Bédard Lab identified that GEM1 transcripts were highly elevated in reversible growth arrested chicken embryo fibroblasts (CEF). In this study we further explore and characterize the protein expression of GEM1 in quiescent CEF. We also design and test shRNAi retroviral constructs to downregulate GEM1 in quiescent CEF.

<p>A microarray analysis of RNA from contact inhibited CEF indicated a hypoxic signature in the contact inhibition program of gene expression (Ghosh <em>et al</em>., 2009). The purpose of this thesis was to investigate whether GAS genes known to be induced during contact inhibition are inducible by hypoxia. The gene p20K was selected as the model for this investigation because it is a growth arrest specific (GAS) gene with a well-characterized promoter (Mao <em>et al</em>., 1993). p20K expression was shown to be positively regulated in hypoxia. It was then determined by transient expression assay that this induction occurred at the promoter level. Interestingly by dissecting the promoter it was found that the quiescent responsive unit (QRU) was required for promoter induction during hypoxia. It has previously been shown that the QRU was required for contact inhibition induction of p20K in a C/EBPβ dependent manner (Mao <em>et al</em>., 1993; Kim <em>et al</em>., 1999).</p> <p>The mechanism behind hypoxic induction of the QRU was then investigated. The kinetics of HIF1α and p20K induction during hypoxia demonstrated that HIF1α was transiently expressed between 2-8 hrs of hypoxia while p20K was induced after 8 hrs of hypoxia. Co-Immuniprecipitation assay was also used to determine if a HIF1α-C/EBPβ interaction occurred, however, this molecular interaction could not be shown. These experiments suggests that HIF1α is not involved with the induction of the QRU. Over-expression of the dominant negative C/EBPβΔ184 repressed p20K induction, thus implicating C/EBPβ in activation in both contact inhibition and hypoxia. We also observed by western blot analysis that the C/EBP family member CHOP was repressed during hypoxia, causing a decrease in the amount of CHOP-C/EBPβ complexes in the cell. It was also found that over-expression of CHOP antagonized the induction of p20K by hypoxia. In conclusion hypoxia represses CHOP levels resulting in an increase of potent C/EBPβ homodimers at the expense of the inactive CHOP-C/EBPβ heterodimers.</p> / Master of Science (MSc)

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Conditions which promote reversible growth arrest, such as hypoxia and high cell density, lead to activation of a diverse network of proteins known as growth arrest specific (GAS) genes. Fatty acid binding protein 4 (FABP4), a lipid chaperone involved in the regulation of metabolic and inflammatory responses, has been shown to be part of the GAS program. While the induction of FABP4 in oxygen-deprived environments is well characterized, its functionality and regulation in such conditions remains unclear. In this study, we describe how mis-expression of FABP4 affects cell viability and survival within low oxygen conditions. Loss of FABP4 using shRNA was shown to be associated with a significant increase in oxidative stress and lipid peroxidation, a reduction in lipid droplet formation and a greater incidence of apoptosis. Hypoxia-mediated expression of FABP4 was also found to be positively correlated with cellular levels of C/EBP-beta, an essential activator of p20K in quiescence. FABP4 and p20K are both lipocalins that have been shown to share similar induction patterns and ability to assist in the maintenance of lipid trafficking in cellular stress circumstances. Unexpectedly, the depletion of FABP4 or p20K results in loss of the other in limited oxygen concentrations. This occurs independently of disruption to the broad GAS gene program, suggesting the two proteins may be co-regulated in a shared hypoxic-signalling pathway. C/EBP-beta appears to be the transcriptional activator shared by FABP4 and p20K in quiescence, and the three may be part of an intricate system to sense and respond to reactive oxygen species and lipid radicals. However, the forced expression of either FABP4 or p20K when the other is repressed only moderately restores cell survival through alleviating oxidative stress, indicating the two are both necessary for optimal response to hypoxia. In all, these studies suggest that analogous to the p20K lipocalin, FABP4 plays a critical role in lipid homeostasis and cell survival in conditions of limited oxygen concentrations, and its stimulation is dependent on C/EBP-beta activity. / Thesis / Master of Science (MSc) / A study investigating the role of FABP4 and p20K in conditions of reversible growth arrest with an emphasis on cell survival, lipid homeostasis and mitigating the effects of oxidative stress, and regulation of the two lipocalins by C/EBP-beta.