Signal Transducer and Activator of Transcription 3 Activates CCAAT Enhancer-binding Protein d Gene Transcription in G0 Growth-arrested Mouse Mammary Epithelial Cells and in Involuting Mouse Mammary Gland*

The CCAAT enhancer-binding protein (C/EBP) family of transcription factors is implicated in the regulation of cell proliferation and differentiation in a variety of tissues. C/EBPd is involved in regulating G0 growth arrest and apoptosis of mouse mammary epithelial cells. This study shows that activation of signal transducer and activator of transcription 3 (Stat3), but not activation of Stat1 or Stat5, occurs concurrently with G0 growth arrest of HC11 mouse mammary epithelial cells, but not NIH 3T3 fibroblasts. Promoter analysis demonstrates that the C/EBPd promoter fragment involved in transcriptional activation during G0 growth arrest contains a Stat3 binding site and that mutation of this site eliminates the G0 growth arrest inducibility of the C/EBPd promoter. Overexpression of Stat3 increases C/EBPd promoter activity during G0 growth arrest of HC11 cells, whereas dominant negative Stat3 decreases C/EBPd promoter activity under the same conditions. Neither Stat3 overexpression nor dominant negative Stat3 expression influences C/EBPd promoter activity in growing HC11 cells or G0 growth-arrested NIH3T3 cells, demonstrating that the effect is specific to G0 growth arrest of mammary epithelial cells. Band shift assays and antibody interference assays demonstrate specific binding of Stat3 to the acute phase response element in the C/EBPd promoter in G0 growth-arrested HC11 cell extracts and 24 h involuting mouse mammary gland extracts. These data indicate that Stat3 activates C/EBPd transcription in G0 growth-arrested mouse mammary epithelial cells and binds to the C/EBPd promoter during involution. An autocrine mechanism of Stat3 activation is proposed.