Retinoic acid differentially regulates retinoic acid receptor-mediated pathways in the HEP3B cell line

Retinoic acid (RA) up-regulates retinoic acid receptor β (RARβ) gene expression in a variety of cell lines. Whether up-regulation of the RARβ gene reflects increased activity in a RARβ-mediated biological process is unclear since RARβ tends to heterodimerize with retinoid x receptor (RXR). In F9 teratocarcinoma cell line, RA-induced differentiation is accompanied by increased expression of the RARE, RXRα, and α-fetoprotein (AFP) genes. Previously, we have shown that the RA-mediated regulation of the AFP gene is through RXRα homodimers. In contrast to F9 cells, Hep3B is unique in that the AFP gene is down-regulated by RA in a manner reminiscent of down- regulation of AFP in postfetal liver. In this paper, we have examined the RA- mediated regulation of the RAR, RXR, peroxisome proliferator-activated receptor (PPAR), and AFP genes in Hep3B cells. RA induced the expression of RARα, β, and γ mRNA in Hep3B cells. However, the expression of RXRα mRNA was down-regulated, and the levels of RXRβ and RXRγ mRNA remained unchanged after RA treatment. In addition, the expression of the PPARα, β, and γ genes was also unchanged. Gel retardation assays demonstrated that RA decreased the overall binding of nuclear receptors to the RA and PPAR response elements. By super-shift assays using specific anti-RAR and -RXR antibodies, RA treatment decreased the amount of RXRα while increasing the amount RARE bound to retinoic acid response element-DR1 (direct repeat with spacer of one nucleotide), indicating the levels of RAR/RXR heterodimer, RXR/RXR homodimer, or RAR/RAR homodimers were altered upon RA treatment of Hep3B cells. In addition, the RA-mediated reduction of RXRα in part results in down-regulation of the AFP gene. Our data indicates that RA exerts its effects by differentially regulating its own receptor gene expression.