Progesterone (P) and its receptor, the progesterone receptor (PR), are important for mammary gland development. Moreover, P/PR signaling also contributes to mammary tumorigenesis. Thus, studying the mechanism of PR expression is important in breast cancer research. C/EBPB-deficient mice and mice blocked for AP-1 activity show similar defects in mammary gland development as do PRB-deficient mice, especially during pregnancy, suggesting that these transcription factors might act in the same pathway or may regulate overlapping sets of downstream target genes. An overall decrease in PR observed in sexually mature wild type mice fails to occur in C/EBPB-deficient mice, while no alterations in C/EBPB expression are observed in PR-deficient mice. Moreover, AP-1 has been found to regulate PR expression. These observations suggest that C/EBPB and AP-1 act upstream of PR. This leads us to study the possibility that C/EBPB and AP-1 are required for PR expression.We examined whether C/EBPB participated in the transcriptional regulation of PR expression in the mammary gland. Transient co-transfection of a PR promoter-reporter construct with expression vectors that individually express C/EBPB isoforms (LAP1, LAP2, or LIP) into a mouse mammary carcinoma cell line revealed that all C/EBPB isoforms, surprisingly including LIP (the shortest isoform lacking transactivation domains), can transactivate the PR promoter. Importantly, we found that LIP, in particular, robustly synergizes with an AP-1 member, c-Jun, to drive PR transcription. Consistent with significant roles for C/EBPB and c-Jun in PR expression, knockdown experiments showed that endogenous levels of C/EBPB and c-Jun expression were sufficient to drive the PR promoter-reporter. Additionally, overexpression of LIP elevated PR protein expression from the intact endogenous gene encoding PR. Furthermore, in vivo immunofluorescence studies showed that C/EBPB and PRA expression are mutually exclusive in the mammary epithelium, while PRB is only expressed in cells that express C/EBPB. This suggests an important role for C/EBPB in PRB expression during pregnancy. Then, we studied the mechanism by which LIP and c-Jun synergistically activate the PR promoter. We demonstrated in the reporter assay that the integrity of C/EBP- and AP-1-binding sites was required for the respective C/EBPB; and c-Jun activities on the PR promoter. Moreover, we showed in ChIP assay that efficient promoter occupancy of both LIP and c-Jun and their synergistic transactivation of the PR promoter required at least one C/EBP- and one AP-1-binding site. In addition, as indicating in the sequential ChIP assay, C/EBPB and c-Jun simultaneously occupied PR promoter. This leads us to propose a model where the synergy of C/EBPB and c-Jun in transactivation of the PR promoter is dependent on the two factors mutually stabilizing their recruitment to the PR promoter. Collectively, our data suggest a critical role for C/EBPB, particularly LIP, in PRB expression.
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