RXR-MEDIATED REGULATION OF THE ALPHA-FETOPROTEIN GENE THROUGH AN UPSTREAM ELEMENT

Retinoic acid (RA) is known to have potent effects on development and differentiation. RA exerts its effects on transcription through two di stinct classes of nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR), that bind to specific RA-responsive elements (RARE) in target genes, alpha-Fetoprotein (AFP), a hepatocyt e differentiation, maturation, and carcinogenesis marker, is transcrip tionally upregulated by RA in McA-RH8994 hepatoma cells, Using deletio n mapping analysis, we have identified a RARE-like sequence that is lo cated between -2406 and -2378 of the transcription initiation site of the rat AFP gene, Sequence analysis demonstrated that this cis-acting element consists of three direct repeats and one inverted repeat of a GGGTCA-like half-site, The putative RARE can specifically bind to both RXR homodimers and RAR/RXR heterodimers as determined by gel mobility shift assays, A DR1 direct repeat was more efficient than a DR5 direc t repeat oligonucleotide in competition for binding of the putative RA RE to RXR and RAR/RXR, A mutagenesis study indicated that to have a fu ll-strength induction. all the repeats were required, To further analy ze the function of this element in vivo, a reporter gene construct of the putative RARE combined with the thymidine kinase promoter was cotr ansfected with RAR and RXR expression plasmids in CV1 cells, CAT assay s demonstrated that overexpression of RXR alpha conferred the best RA response, consistent with our previous observation that 9-cis-RA is mo re potent than all-trans-RA for inducing the expression of the AFP gen e, In addition, the RXR selective ligand LG100153 alone can stimulate the expression of the AFP gene, Our data suggest that an RXR-mediated pathway exists for modulation of AFP gene expression through a specifi c element.