Mechanisms underlying the induction of the putative human tumor suppressor GPRC5A by Retinoic acid

Abstract

Retinoic acid regulates the expression of genes involved in cell proliferation,

differentiation, and survival by direct control of gene transcription via activation of

nuclear retinoid receptors bound to response elements in the promoters of target genes

or by indirect mechanisms. Herein, we investigated the mechanism by which retinoic

acid induces the expression of the human tumor suppressor GPRC5A. The proximal 5’

upstream region of the GPRC5A gene was found to contain two potential RAR/RXR

binding sites (RAREs) and one VDR/RXR binding site with direct repeat 5 (DR5) motifs

designated DR5I (-489 to -473), DR5II (-136 to -120), and DR5III (-81 to -65). DR5II and

DR5III but not DR5I were conserved among vertebrates. However, only DR5III (5’-

TGTCCCTCTGCTCACCC-3’) was found to be the functional RARE for mediating

induction of GPRC5A as indicated by electrophoretic mobility shift assay using wild type

and mutated synthetic oligonucleotides representing different fragments of the promoter

for competition with retinoic acid receptor β RARE. Chromatin immunoprecipitation

assay confirmed the binding of retinoic acid receptors α and γ and retinoid X receptors α

and β to DR5III in intact cells. These results demonstrate the importance of functional

analysis for validating the activity of predicted response elements.