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Identification of a Retinoic Acid-Inducible Element in the Murine PTH/PTHrP (Parathyroid Hormone/Parathyroid Hormone-Related Peptide) Receptor Gene

Department of Endocrinology (M.K., H.F.-S., S.E.P., C.W.G.M.L.) and Department of Pediatrics (M.K.) Leiden University Medical Center 2300 RC Leiden, The Netherlands
Netherlands Institute for Developmental Biology (J.A.A.H., L.H.K.D.) 3584 CT Utrecht, The Netherlands
Endocrine Unit (B.L., A.-B.A.-S.) Massachusetts General Hospital Boston, Massachusetts 02114

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 inducer of extraembryonic endoderm formation and PTH/PTHrP-receptor expression in embryonal carcinoma (EC) and embryonal stem (ES) cells. Using the P19 EC cell line, we have characterized promoter elements of the murine PTH/PTHrP-receptor 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 the downstream P2 promoter. Analysis of promoter reporter constructs in transiently 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 hormone response element consisting of a direct repeat with an interspacing of 1 bp (R-DR1). The R-DR1 efficiently binds retinoic acid receptor- (RAR)-retinoid X receptor- (RXR) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXR heterodimers and RXR and COUP-TFI homodimers in a bandshift assay using extracts of transiently transfected COS-7 cells. RA differentiation of P19 EC cells strongly increases protein binding to the R-DR1 in a bandshift assay. This is caused by increased expression of RXR (, ß, or ) and by the induction of expression of RARß and COUP TFI/TFII, which bind to the R-DR1 as shown by supershifting antibodies. The presence of RXR (, ß, or ) 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 importance 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-induced expression of PTH/PTHrP-receptor promoter reporter constructs. Since this mutation does not completely abolish RA-induced expression of PTH/PTHrP-receptor promoter reporter constructs, sequences other than the R-DR1 might also be involved in the RA effect. Finally, we show that the RA-responsive promoter region is also able to induce expression of a reporter gene in extraembryonic endoderm of 7.5 day-old transgenic mouse embryos.