Rita Kempf

CCAAT/Enhancer-binding Proteins Are Mediators in the Protein Kinase A-dependent Activation of the Decidual Prolactin Promoter

In the course of decidualization, human endometrial stromal cells (ESC) activate the alternative upstream promoter of the decidual prolactin (dPRL) gene. The dPRL promoter is induced by the protein kinase A pathway in a delayed fashion via the region −332/−270 which contains two overlapping consensus binding sequences, B and D, for CCAAT/enhancer-binding proteins (C/EBP). Here we show that sites B and D both bind C/EBPβ and -δ from ESC nuclear extracts. When decidualization of cultured ESC was induced by treatment with 8-Br-cAMP, complex formation on sites B and D was enhanced. Western blot analysis revealed an elevation of both C/EBPβ isoforms, liver-enriched activator protein and liver-enriched inhibitory protein, with a delayed onset between 8 and 24 h of cAMP treatment, while C/EBPδ expression remained unaffected. Cyclic AMP-mediated activation of dPRL promoter construct dPRL-332/luc3 was abrogated by mutation of sites B and D at −310/−285. An expression vector for liver-enriched activator protein potently induced transcription of dPRL-332/luc3 and further enhanced cAMP-mediated induction, while liver-enriched inhibitory protein expression vector abolished the cAMP response, implying that C/EBPs serve as mediators in the delayed cAMP signal transduction to the dPRL promoter. The ratio between activating and repressing isoforms is likely to dictate the transcriptional output.

Physical Interaction and Mutual Transrepression between CCAAT/Enhancer-binding Protein β and the p53 Tumor Suppressor

The tumor suppressor protein p53 is not only involved in defending cells against genotoxic insults but is also implicated in differentiation processes, a function that it shares with the CCAAT/enhancer-binding protein β (C/EBPβ). We previously reported an up-regulation of both factors in the cycle-dependent differentiation process of human endometrial stromal cells, termed decidualization. C/EBPβ-mediated activation of a decidualization marker, the decidual prolactin promoter, was antagonized by p53. Here we report that C/EBPβ in turn represses the transcriptional activity of p53. Competition for limiting amounts of coactivator CREB-binding protein/p300 was ruled out as the underlying mechanism of transrepression. Physical interaction between p53 and C/EBPβ was demonstrated in vitro and in vivo and shown to depend on the C-terminal domains of both proteins. In gel shift experiments, C/EBPβ reduced complex formation between p53 and its response element. Conversely, p53 strongly inhibited binding of endogenous C/EBPβ from endometrial stromal cells to the C/EBP-responsive region in the decidual prolactin promoter. The observed negative cross-talk between p53 and C/EBPβ is likely to impact expression of their respective target genes.