Characterizing the Role of the Growth Arrest Specific p20K Lipocalin in Chicken Embryo Fibroblasts

Abstract

Cell cycle progression is regulated by complex processes which determine whether cells can continue dividing or enter a nonproliferative state. Cells exit the cell cycle and are able to enter a reversible state of growth arrest called quiescence. A subset of genes involved in mediating cellular quiescence are referred to as growth arrest specific (Gas) genes, however their mechanisms and regulatory characteristics require further analyses. The p20K lipocalin, a Gas gene, is involved in lipid transport is induced upon conditions, such as contact inhibition and hypoxia, in chicken embryo fibroblasts (CEF). The purpose of this study is to characterize the role of the p20K lipocalin upon quiescence in CEF. P20K expression was downregulated in CEF, and the cells were cultured under conditions of contact inhibition and hypoxia. There was a decrease in cell accumulation and an increase in apoptosis in CEF which downregulated p20K when placed in hypoxic conditions. Additionally, there were increased levels of reactive oxygen species (ROS) in hypoxic CEF with p20K downregulation. These results suggest that p20K regulates cellular stress by promoting cell survival and mitigating oxidative stress, however the mechanisms by which these functions are carried out remain unknown. The study also focused on characterizing the relationship between the lipocalins p20K and fatty acid binding protein 4 (FABP4). FABP4 is a Gas gene that is expressed in contact inhibited and hypoxic conditions of quiescence. FABP4 expression was analyzed in CEF with p20K downregulation in conditions of quiescence. The mis-expression of p20K impaired FABP4 expression in contact inhibited and hypoxic CEF, and the ectopic up-regulation of p20K impaired FABP4 expression in contact inhibited CEF. These results suggest that p20K and FABP4 are co-regulated in conditions of contact inhibition and hypoxia. Overall, the p20K lipocalin is involved in cell survival and mitigating oxidative stress in quiescence which can help explain its role in lipid metabolism and the novel “Membrane Stress Response”.