title: Inhibition of 1,25-dihydroxyvitamin D3-dependent transcription by synthetic LXXLL peptide antagonists that target the activation domains of the vitamin D and retinoid X receptors.
abstract: The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through its ability to regulate cellular programs of gene expression. Although RXR appears to participate as a heterodimeric partner with the VDR, absolute evidence for its role remains equivocal in vivo. To test this role and to investigate the requirement for comodulator interaction, we identified VDR- and retinoid X receptor (RXR)-interacting LXXLL peptides and examined whether these molecules could block vitamin D and 9-cis retinoic acid (9-cis RA) response. We used a mammalian cell two-hybrid system to screen a series of nuclear receptor (NR)-reactive LXXLL peptides previously identified through phage display screening for hormone-dependent reactivity with either VDR or RXR. Three categories of peptides were identified: those reactive with both VDR and RXR, those selective for RXR, and those unreactive to either receptor. Peptide fusion proteins were then examined in MC3T3-E1 cells for their ability to block induction of the osteocalcin (OC) promoter by 1,25(OH)2D3 or stimulation of a retinoic acid response element-thymidine kinase (RARE-TK) reporter by 9-cis-RA. Peptides that interacted with both VDR and RXR blocked 1,25(OH)2D3-dependent transcription by up to 75%. Control LXXLL sequences derived from Src-1 and Grip also suppressed 1,25(OH)2D3-induced transactivation; peptides that interacted with RXR blocked 9-cis-RA-induced transcription. Interestingly, two RXR-interacting peptides were also found to block 1,25(OH)2D3 response effectively. These studies support the idea that comodulator recruitment is essential for VDR- and RXR-mediated gene expression and that RXR is required for 1,25(OH)2D3-induced OC gene transcription. This approach may represent a novel means of assessing the contribution of RXR in various endogenous biological responses to 1,25(OH)2D3.
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio, USA.
authors.forename: Peterson
authors.initials: P
authors.lastname: Pathrose
authors.forename: Olga
authors.initials: O
authors.lastname: Barmina
authors.forename: Ching-Yi
authors.initials: CY
authors.lastname: Chang
authors.forename: Donald P
authors.initials: DP
authors.lastname: McDonnell
authors.forename: Nirupama K
authors.initials: NK
authors.lastname: Shevde
authors.forename: J Wesley
authors.initials: JW
authors.lastname: Pike
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002117:Calcitriol / Q000494:pharmacology*; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D011233:Precipitin Tests; D018167:Receptors, Calcitriol / Q000037:antagonists & inhibitors*; D018168:Receptors, Retinoic Acid / Q000037:antagonists & inhibitors*; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000037:antagonists & inhibitors*; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D009842:Oligopeptides; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D002117:Calcitriol