CCAAT/enhancer binding protein δ (C/EBPδ) regulation and expression in human mammary epithelial cells: II. Analysis of activating signal transduction pathways, transcriptional, post‐transcriptional, and post‐translational control

Abstract

CCAAT/enhancer binding protein delta (C/EBPdelta) plays a key role in mammary epithelial cell G0 growth arrest. C/EBPdelta gene expression is down-regulated in rodent mammary tumorigenesis and in human breast cancer, suggesting that "loss of function" alterations in C/EBPdelta gene expression are common in mammary gland malignancies. The goal of this study was to systematically investigate the mechanisms controlling C/EBPdelta gene expression in MCF-10A and MCF-12A human mammary epithelial cell lines. The results demonstrate that G0 growth arrest conditions (i.e., serum and growth factor withdrawal or Oncostatin M (OSM) treatment) result in the activation of JAK1, JAK2, and Tyk 2, members of the Janus kinase family of non-receptor tyrosine kinases, in MCF-10A and MCF-12A cells. Growth arrest or OSM treatment also specifically increases activated (phosphorylated) signal transduction and activators of transcription 3 (STAT3) levels, demonstrating that STAT3, not STAT1 or STAT5, is the downstream target of the activated Janus kinases in MCF-10A and MCF-12A cells. Whole cell lysates from G0 growth arrested (GA) and OSM-treated MCF-12A cells exhibit increased acute phase response element (APRE) binding compared to lysates from growing (GR) MCF-12A cells. Transient transfection using C/EBPdelta promoter-luciferase constructs demonstrated that the APRE (STAT3) consensus binding site is essential for growth arrest or OSM induction of the C/EBPdelta promoter. Mutation of the C/EBPdelta promoter STAT3 site or expression of a dominant negative STAT3 construct (STAT3delta) reduces C/EBPdelta promoter activity in response to growth arrest conditions. The human C/EBPdelta promoter also contains an Sp1 site at -61 bp (relative to the transcriptional start site) which is required for basal transcriptional activation. Mutation or deletion of the Sp1 site decreases promoter activity in response to growth arrest conditions. Treatment with the transcriptional inhibitor actinomycin D demonstrated that the C/EBPdelta mRNA exhibits a relatively short half-life (approximately 40 min). Similarly, treatment with the translational inhibitor anisomysin demonstrated that the C/EBPdelta protein half-life was also relatively short (approximately 160 min). These results indicate that the human C/EBPdelta gene is controlled at multiple levels, consistent with a role for C/EBPdelta in cell cycle control and/or cell fate determination.

Eicosapentaenoic Acid Demethylates a Single CpG That Mediates Expression of Tumor Suppressor CCAAT/Enhancer-binding Protein δ in U937 Leukemia Cells

“…C/EBP␦-enhanced expression is ascribed to demethylation of a site-specific CpG downstream of the Sp1 site in the C/EBP␦ proximal promoter (Fig. 4C) required for C/EBP␦ basal transcriptional activation (58). Indeed, methylation of this specific CpG impaired Sp1 binding to its consensus sequence (Fig.…”

Promoter methylation reduces C/EBPδ (CEBPD) gene expression in the SUM-52PE human breast cancer cell line and in primary breast tumors

“…In contrast, human breast cancer cell lines (MDA-MB-231, T-47D, Skbr-3 and MCF-7) exhibit reduced C/EBPd gene expression and impaired growth inhibition in response to growth arrest treatments [21][22][23][24]. To further investigate C/EBPd gene expression we investigated CEBPd gene expression in the SUM-44PE and SUM-52PE human breast cancer cell lines developed at the University of Michigan [31].…”

“…STAT3 activation (phosphorylation, pSTAT3), dimerization, nuclear translocation and DNA binding are key biochemical events in the transcriptional activation of CEBPd gene expression in response to GA and IL-6 family cytokine treatment in hepatocytes and mammary epithelial cells [21][22][23][24]32]. To determine if STAT3 is altered in SUM breast cancer cell lines we investigated STAT3 levels, STAT3 activation (phosphorylation, pSTAT3) and C/EBPd protein levels.…”

“…'Loss of function' alterations in C/ EBPd gene expression have been specifically identified in primary human breast tumors and in experimental mammary tumorigenesis [15][16][17]. Our laboratory has reported that C/EBPd gene expression is highly induced in mouse and human mammary epithelial cells in response to growth arrest treatments including: serum and growth factor withdrawal, contact inhibition and IL-6 family cytokine treatment [18][19][20][21][22][23][24]. We directly assessed the growth suppressor function of C/EBPd in vitro and found that over expression of C/EBPd accelerates mammary epithelial cell growth arrest (G 0 entry) and that antisense reduction in C/EBPd gene expression delays mouse mammary epithelial cell growth arrest (G 0 entry) [19].…”

“…In vivo, virgin female CEBPd knockout mice exhibit excessive mammary ductal branching due to persistent, low level proliferation, a possible consequence of defective G 0 growth arrest [25]. In vitro studies with both mouse and human mammary epithelial cells revealed that signal transducer and activator of transcription3 (STAT3) and Sp1 play key roles in the growth arrest induction of CEBPd gene expression [21][22][23][24].…”