Retinoids are known to modulate macrophage differentiation, proliferation, and function including cytokine gene expression. However, signaling of retinoic acid (RA), a biologically active metabolite of vitamin A, in Kupffer cells has not been characterized. This study reports mRNA expression by rat Kupffer cells of RA receptor (RAR) and retinoid X receptor (RXR) subtypes and their binding activities to the RA responsive element (RARE) or retinoid X responsive element (RXRE). Total RNA and nuclear proteins were extracted from Kupffer cells immediately following isolation from livers of normal male Wistar rats. Competitive PCR demonstrated relative mRNA expression of RAR and RXR subtypes in the order of beta>alpha>gamma for and alpha>beta>gamma, respectively. It also demonstrated that the RXR alpha and beta mRNA levels were 5- to 10-fold higher in Kupffer cells than in hepatic stellate cells while RAR mRNA expression was shown to be similar for all the subtypes in both cell types. Gel mobility shift assays of nuclear extracts with labeled RARE and RXRE probes showed distinct binding activities for both responsive elements, which were effectively displaced with cold probes in excess but not with an unrelated oligonucleotide. A supershift assay with an antibody against RARalpha or RXRalpha has confirmed the contribution of both receptors to RARE binding and that of the RXRalpha to RXRE binding activity. These results represent the first demonstration of RA signaling at the nuclear level in Kupffer cells.
Copyright 2000 Academic Press.