CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: II. Analysis of activating signal transduction pathways, transcriptional, post-transcriptional, and post-translational control.

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.