Characterizing The Transcriptional Regulation And Role Of The Growth Arrest Specific P20K Lipocalin In Chicken Embryo Fibroblasts

Abstract

The p20K lipocalin was originally identified to be expressed in chicken heart mesenchymal cells entering growth arrest in response to high cell density. Preliminary studies analyzing the transcriptional activation of p20K within growth arrest revealed that it is predominantly controlled by C/EBPb, which binds to two regions within the p20K promoter (QRU). Although the transcriptional activation of p20K has been thoroughly examined, it has yet to be defined how p20K is regulated in cycling and starved states. In this study, we observe that C/EBPb and the MEK pathway are the dominant regulators of p20K expression. This regulation was mutually exclusive, as C/EBPb was found to associate with the QRU and promote p20K expression during states of growth arrest, whereas the serine/threonine kinase ERK2 bound and repressed p20K expression in cycling states. Furthermore, we show that the ER stress includible protein CHOP acts to block p20K expression in states of ER stress or starvation through the interaction with C/EBPb. Transcriptome analysis done in conjunction with these studies revealed that many hypoxia-responsive specific genes, including carbonicanhydrase IX, were expressed within states of contact inhibition. These studies were supported when nitroreductase activity, a marker of hypoxia, was detected in cells at confluence. These results suggest that cells at high density experience hypoxia, and as such, p20K may be induced in a hypoxia-specific manner. Studies analyzing the function of p20K in hypoxia revealed that the knockdown of p20K via shRNA negatively affected cell viability within low oxygen concentrations. This was shown to be associated with a significant increase in lipid per oxidation and lipid accumulation within hypoxia, and ultimately resulted in a higher incidence of apoptotic cells. In all, these studies suggest that p20K plays a critical role in lipid homeostasis and cell survival within conditions of limited oxygen concentrations.