Please use this identifier to cite or link to this item:
|Title:||CHOP IS A KEY REGULATOR OF p20K IN HYPOXIA AND CONTACT INHIBITED CHICKEN EMBRYONIC FIBROBLASTS|
|Authors:||Camacho, Dora Y.|
|Abstract:||<p>Growth arrest specific (GAS) genes are responsible for regulating cell proliferation, gene expression, and apoptosis (Fleming et al., 1997). Given these roles, it is important to study the mechanisms of action and regulation of Gas genes in quiescence (G<sub>0</sub>). If altered, quiescence can lead to abnormal development, degenerative diseases, and cancer (Liu et al., 2007). The p20K lipocalin is a GAS gene expressed in contact inhibited cells (Kim et al., 1999). Recent studies have also demonstrated that p20K is highly inducible in hypoxia, and its activation in hypoxia and contact inhibition is dependent upon a 48bp promoter region referred to as the <em>Quiescence Responsive Unit</em> (QRU) (Mao et al. 1993; S. Kim et al. 1999; Fielding, MSc. Thesis, 2011). Furthermore, p20K is highly regulated by <em>CAAT/enhancer binding protein</em> (C/EBPβ) as C/EBPβ crucial interaction with different QRU binding sites are essential for the activation of p20K (Kim et al., 1999). CHOP, a C/EBP homologous protein belonging to the C/EBP family, has been identified as an inhibitor of C/EBPβ activity (Sok et al., 1999). Co-immunoprecipitation experiments showed that CHOP induction either by ER stress or starvation promoted the formation of C/EBPβ heterodimers inhibiting p20K expression in Chicken Embryonic Fibroblasts (CEFs). Experiments using a CHOP knockdown by shRNA demonstrated that CHOP repression induced robust expression of p20K in cycling CEF cells after 24 hours in hypoxia, but not at earlier time points. In addition, as documented by RT-qPCR and Western blotting analysis, both p20K and CHOP mRNA and protein expression levels show to have an inverse relationship in hypoxia and contact inhibition. Since CHOP expression is greatly induced during ER stress and starvation, two ER stress inducible agents (tunicamycin and thapsigargin) were used to stimulate high levels of CHOP in CEF, leading to the induction of CHOP and repression of p20K. CEF were also treated with DMOG, a hypoxia mimetic that can stabilize Hypoxia Inducible Factor (HIF) under normoxic conditions (Barnucz et al., 2013). Surprisingly, in DMOG treated cells CHOP levels did not decrease, while p20K was highly induced. At later time points, while DMOG induces modest induction of CHOP, it decreases phospho-ERK levels (a transcriptional repressor of p20K) demonstrating a separate signal/pathway controlling p20K induction in these conditions that requires follow up studies. Collectively, these results demonstrate that the down-regulation of the p20K GAS gene in response to ER stress is mediated by the induction of CHOP and segregation of C/EBPβ as heterodimers.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
Files in This Item:
|fulltext.pdf||2.12 MB||Adobe PDF||View/Open|
Items in MacSphere are protected by copyright, with all rights reserved, unless otherwise indicated.