ISOFORM-SPECIFIC RETINOID-X RECEPTOR (RXR) ANTIBODIES DETECT DIFFERENTIAL EXPRESSION OF RXR PROTEINS IN THE PITUITARY-GLAND

There are three known isoforms of the retinoid-X receptor (RXR): RXR a lpha, RXR beta, and RXR gamma. RXR alpha and RXR beta messenger RNAs a re widely expressed, whereas RXR gamma messenger RNA is restricted to only a few tissues, including embryonic pituitary gland. Little is kno wn about the level of expression and cell distribution of RXR proteins in the adult pituitary gland. To examine these issues further, we rai sed isoform-specific polyclonal antibodies against each of the known m ouse RXR isoforms using synthetic peptides containing isoform-specific epitopes from the amino-terminal region. The specificity of each anti body was confirmed by immunoprecipitation, Western immunoblot analysis , and electrophoretic mobility shift assay with supershift studies of in vitro translated RXR isoforms. Immunocytochemical analysis showed t hat anti-RXR alpha and anti-RXR beta antisera stained the nuclei of mo st pituitary cells. In contrast, anti-RXR gamma antiserum stained the nuclei of only a few cells throughout the pituitary. In the hypothyroi d 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 pitu itary with antibodies against pituitary hormones indicated that RXR ga mma protein was predominantly expressed in thyrotropes. Antibody super shift experiments using nuclear extracts of adult rat whole pituitary and rodent pituitary cell lines showed that anti-RXR gamma antibody co uld alter the mobility of protein-DNA complexes formed only from nucle ar extracts of rat whole pituitary and thyrotropic TtT-97 cells. In co ntrast, anti-RXR alpha and anti-RXR beta antibodies could supershift p rotein-DNA complexes formed from nuclear extracts of all cell Lines te sted. RXR gamma protein expression in TtT-97 cells also was observed b y Western immunoblot analyses. Therefore, there is thyrotrope-predomin ant expression of RXR gamma protein. We speculate that RXR gamma may p lay a role in the regulation of thyroid hormone target genes in thyrot ropes and possibly cell type differentiation in the pituitary.