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 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-alpha (RARalpha)-retinoid X receptor-alpha (RXRalpha) and chicken ovalbumin upstream promoter (COUP)-transcription factor I (TFI)-RXRalpha heterodimers and RXRalpha 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 band-shift assay. This is caused by increased expression of RXR (alpha, beta, or gamma) and by the induction of expression of RARbeta 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 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.
Karperien
M
M
Department of Endocrinology, Leiden University Medical Center, The Netherlands. karperien@rullf2.leidenuniv.nl
Farih-Sips
H
H
Hendriks
J A
JA
Lanske
B
B
Papapoulos
S E
SE
Abou-Samra
A B
AB
Löwik
C W
CW
Defize
L H
LH
D000818:Animals; D001483:Base Sequence; D051836:COUP Transcription Factors; D018236:Carcinoma, Embryonal / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D004707:Endoderm / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010281:Parathyroid Hormone / Q000235:genetics* / Q000378:metabolism; D044162:Parathyroid Hormone-Related Protein; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011987:Receptors, Steroid; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D020218:Response Elements / Q000502:physiology*; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014212:Tretinoin / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D018168:Receptors, Retinoic Acid; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; C082925:retinoic acid receptor beta; D014212:Tretinoin; D001616:beta-Galactosidase