EXPRESSION OF THE PARATHYROID HORMONE-RELATED PEPTIDE GENE IN RETINOIC ACID-INDUCED DIFFERENTIATION - INVOLVEMENT OF ETS AND SP1

Differentiation of P19 embryonal carcinoma (EC) and embryonal stem (ES )-5 cells with retinoic acid (RA) induces expression of PTH-related pe ptide (PTHrP) mRNA. In this study we have characterized a region betwe en nucleotide (nt) -88 and -58 relative to the transcription start sit e in the murine PTHrP gene that was involved in this expression, Seque nce analysis identified two partially overlapping binding sites for th e Ets family of transcription factors and an inverted Sp1-binding site . Two major specific bands were detected in a bandshift assay using an oligonucleotide spanning nt -88 and -58 as a probe and nuclear extrac ts from both undifferentiated and RA-differentiated P19 EC cells, The lower complex consisted of Ets-binding proteins as demonstrated by com petition with consensus Ets-binding sites, while the upper complex con tained Sp1-binding activity as demonstrated by competition with consen sus Sp1-binding sites. The observed bandshift patterns using nuclear e xtracts of undifferentiated or RA-differentiated P19 cells were indist inguishable, suggesting that the differentiation-mediated expression w as not caused by the induction of expression of new transcription fact ors, Mutations in either of the Ets-binding sites or the Sp1-binding s ite completely abolished RA-induced expression of PTHrP promoter repor ter constructs, indicating that the RA effect was dependent on the sim ultaneous action of both Ets-and Sp1-like activities. Furthermore, the se mutations also abolished promoter activity in cells that constituti vely expressed PTHrP mRNA, suggesting a central role for the Ets and S p1 families of transcription factors in the expression regulation of t he mouse PTHrP gene.