Characterization of C/EBPdelta mRNA stability regulation in mouse mammary epithelial cell.

摘要

CCAAT/Enhancer binding proteins (C/EBPs) are a family of highly conserved transcription factors that regulate gene expression involved in cellular physiological process including cell growth, differentiation and apoptosis. C/EBPdelta, a C/EBP family member, plays an important role in the G0 growth arrest of mouse mammary epithelial cells in vitro and of mouse mammary gland involution in vivo. Previous reports from our laboratory demonstrated that C/EBP delta gene transcription was highly induced during G0 growth arrest, but that the mRNA was highly unstable (t1/2 = ∼40 minutes). AREs (i.e. AU rich elements) are well-known mRNA destabilizing elements. AREs are present in the 3'UTR of C/EBP delta mRNA, but the underlying mechanism in the regulation of its mRNA stability remained uncharacterized. The goal of this dissertation was to fulfill this unmet need by identifying the regulators that can affect C/EBPdelta expression.; The first purpose of this dissertation was to investigate the mechanism for posttranscriptional regulation of C/EBPdelta gene expression in G 0 growth arrested mammary epithelial cells. Previous studies from our lab demonstrated that C/EBPdelta gene expression was highly induced in response to growth arrest conditions (i.e. serum and growth factor withdrawal, contact inhibition, or cytokine treatment). Using a novel experimental system, I demonstrated that C/EBPdelta mRNA was stabilized in G0 growth arrest after genotoxic stress such as UV or MMS treatment. p38 MAPK activation was responsible for the induced mRNA stabilization. Using a beta-globin transcript reporter, I further demonstrated that the stabilization of C/EBPdelta mRNA was partially mediated through its 3'UTR. HuR, a RNA stabilizing factor, increased its binding to the 3'UTR of C/EBPdelta mRNA after UV treatment, and the cytoplasmic translocation of HuR was dependent on the p38 MAPK pathway. SiRNA analysis confirmed that HuR was required for the induced stabilization of C/EBPdelta mRNA after UV irradiation. Finally, functional analysis of C/EBPdelta expression under UV irradiation revealed that increased expression of C/EBPdelta was associated with apoptosis. Overall, this work characterized the regulatory mechanism for C/EBPdelta mRNA stability in G0 growth arrest under genotroxic stress and provided a better understanding of the role of C/EBPdelta in G0 growth arrest.; The second purpose of this dissertation was to investigate the role of CREB in the transcriptional regulation of C/EBPdelta in G0 growth arrested mouse mammary epithelial cells. Previous studies using nuclear run-on assay and Northern Blot analysis demonstrated that the transcription of C/EBPdelta was highly induced during G0 growth arrest. Results from C/EBPdelta promoter-luciferase analyses and electromobility shift assays demonstrated that activation of STAT3 and binding of STAT3 and Sp1 to the mouse C/EBPdelta promoter were important in the transcriptional activation of C/EBPdelta after serum and growth factor withdrawal and Oncostatin M (OSM) addition. In this dissertation, I demonstrated that CRE site in the proximal promoter of C/EBPdelta was also important for its transcriptional activation under G0 growth arrest. Mutation of the CRE site decreased C/EBPdelta promoter activation. Using ChIP assay, I found that CREB was constitutively bound to the proximal promoter of C/EBPdelta and promoter-bound CREB was phosphorylated after serum and growth factor withdrawal and/or OSM addition. Further study showed that ERK pathway was activated by OSM in a short period of time. The Blockage of CREB phosphorylation by U0126, a MEK inhibitor, inhibited the C/EBP delta expression. Alternatively, repression of CREB by SiRNA led to reduced expression of C/EBPdelta in G0 growth arrest. This work demonstrated besides Stat3 and Sp1, CREB can regulate C/EBPdelta expression, but to a lesser extent.; In summary, this dissertation provided a better understanding of the regulation of C/EBPdel