Identification of a retinoic acid-inducible element in the murine PTH PTHrP (parathyroid hormone parathyroid hormone-related peptide) receptor gene

We have shown previously that the PTH/PTHrP (PTH-related peptide) receptor mRNA becomes expressed very early in murine embryogenesis, i.e. during the formation of extraembryonic endoderm. Retinoic Acid (RA) is a potent induce r of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC c ell line, we have characterized promoter elements of the murine PTH/PTHrP-r eceptor gene that are involved in this RA-induced expression. The data show that RA-induced expression of the PTH/PTHrP-receptor gene is mediated by t he downstream P2 promoter. Analysis of promoter reporter constructs in tran siently transfected P19 cells treated with RA identified an enhancer region between nucleotides -2714 and -2702 upstream of the P2 transcription start site that is involved in the RA effect. This region matches a consensus ho rmone response element consisting of a direct repeat with an interspacing o f 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor-alpha (R AR alpha)-retinoid X receptor-alpha (RXR alpha) and chicken ovalbumin upstr eam promoter (COUP)-transcription factor I (TFI)-RXR alpha heterodimers and RXR alpha and COUP-TFI homodimers in a bandshift assay using extracts of t ransiently transfected COS-7 cells. RA differentiation of P19 EC cells stro ngly increases protein binding to the R-DR1 in a bandshift assay. This is c aused by increased expression of RXR (alpha, beta, or gamma) and by the ind uction of expression of RAR beta and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (alpha, beta, or gamma) in the complexes binding to the R-DR1 suggests that RXR homodimers are involved in RA-induced expression of the PTH/PTHrP-receptor gene. The i mportance of the R-DR1 for RA-induced expression of PTH/PTHrP-receptor was shown by an inactivating mutation of the R-DR1, which severely impairs RA-i nduced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTH rP-receptor promoter reporter constructs, sequences other than the R-DR1 mi ght also be involved in the RA effect. Finally, we show that the RA-respons ive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.