Glucocorticoid (GC) signalling influences the response of the cell to a number of other signals via a mechanism referred to as ‘cross-talk'. This cross-talk may act at several levels, including an interaction between the transcription factors involved in the signalling pathways. In the present paper, we demonstrate a novel functional interaction between GC and all-trans-retinoic acid (RA) signalling. We show that, in P19 embryonal carcinoma cells, GCs potentiate RA-induced expression of the murine Hoxb-1 gene through an autoregulatory element, b1-ARE, recognized by the Pbx1 and HOXB1 homoeodomain proteins. The synergistic effect of GC did not involve GC receptor (GR) binding to the b1-ARE, and the GC—GR complex alone was unable to activate transcription via the element. Furthermore, the ability of the GR to transactivate was not required, excluding expression of a GC-induced protein as the mechanism for the GC/RA synergy. Additional transfection experiments showed that the Pbx1/HOXB1 heterodimer was the target for the GC effect. Furthermore, functional dissection of the GR demonstrated that the DNA-binding domain (DBD) of the GR was required for the synergy. A physical interaction between the GR and Pbx1 proteins was demonstrated in vivo by co-immunoprecipitation experiments. These results are compatible with a model in which the GC/RA synergy is mediated by a direct interaction between the GR and Pbx1. On the basis of the ubiquitous expression of both GR and Pbx1, a number of genes regulated by Pbx are likely to be important targets for GC-mediated ‘cross-talk'.
Abbreviations used: b1-ARE, Hoxb-1 gene autoregulatory element; DBD, DNA-binding domain; Dex, dexamethasone; DTT, dithiothreitol; EMSA, electrophoretic mobility-shift assay; GC, glucocorticoid; GR, GC receptor; GRE, GC-responsive element; NF-κB, nuclear factor κB; NF-IL6, nuclear factor for interleukin 6; nGRE, negative GRE; Oct-1, octamer transcription factor-1; P19 EC cells, P19 embryonal carcinoma cells; R3, sequence motif repeat 3 (a subregion of b1-ARE); RA, all-trans-retinoic acid; RARE, RA-response element; TALE, three-amino-acid loop extension; TR, thyroid hormone receptor.
Present address: Muscle Development Unit, Children's Medical Research Institute, Wentworthville, NSW 2145, Australia.