Isoform-specific retinoid-X receptor (RXR) antibodies detect differential expression of RXR proteins in the pituitary gland.

Abstract

There are three known isoforms of the retinoid-X receptor (RXR): RXR alpha, RXR beta, and RXR gamma. RXR alpha and RXR beta messenger RNAs are widely expressed, whereas RXR gamma messenger RNA is restricted to only a few tissues, including embryonic pituitary gland. Little is known about the level of expression and cell distribution of RXR proteins in the adult pituitary gland. To examine these issues further, we raised isoform-specific polyclonal antibodies against each of the known mouse RXR isoforms using synthetic peptides containing isoform-specific epitopes from the amino-terminal region. The specificity of each antibody was confirmed by immunoprecipitation, Western immunoblot analysis, and electrophoretic mobility shift assay with supershift studies of in vitro translated RXR isoforms. Immunocytochemical analysis showed that anti-RXR alpha and anti-RXR beta antisera stained the nuclei of most pituitary cells. In contrast, anti-RXR gamma antiserum stained the nuclei of only a few cells throughout the pituitary. In the hypothyroid state, however, a marked increase in both the number and density of RXR gamma-immunostained nuclei were observed compared to those in the euthyroid state. Double immunostaining studies of hypothyroid rat pituitary with antibodies against pituitary hormones indicated that RXR gamma protein was predominantly expressed in thyrotropes. Antibody supershift experiments using nuclear extracts of adult rat whole pituitary and rodent pituitary cell lines showed that anti-RXR gamma antibody could alter the mobility of protein-DNA complexes formed only from nuclear extracts of rat whole pituitary and thyrotropic TtT-97 cells. In contrast, anti-RXR alpha and anti-RXR beta antibodies could supershift protein-DNA complexes formed from nuclear extracts of all cell lines tested. RXR gamma protein expression in TtT-97 cells also was observed by Western immunoblot analyses. Therefore, there is thyrotrope-predominant expression of RXR gamma protein. We speculate that RXR gamma may play a role in the regulation of thyroid hormone target genes in thyrotropes and possibly cell type differentiation in the pituitary.