Journal
JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Issue 12, Pages 2196-2205Publisher
WILEY
DOI: 10.1359/jbmr.2002.17.12.2196
Keywords
peptides; transcription; vitamin D receptor; retinoid X receptor; osteocalcin; comodulators; LXXLL motifs
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Funding
- NCI NIH HHS [CA 90645] Funding Source: Medline
- NIDDK NIH HHS [DK-52453] Funding Source: Medline
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The vitamin D receptor (VDR) is known to mediate the biological actions of 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] 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)(2)D-3-dependent transcription by up to 75%. Control LXXLL sequences derived from Src-1 and Grip also suppressed 1,25(OH)(2)D-3-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)(2)D-3 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)(2)D-3-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)(2)D-3.
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