Androgen binding in nuclear matrix of human genital skin fibroblasts from patients with androgen insensitivity syndrome

Abstract

Specific sex steroid-binding sites are associated with the salt-insoluble nuclear matrix from which lipids, histones, and chromatin have been extracted. In intact cultured normal human genital skin fibroblasts incubated for 1 h at 37 C with a saturating concentration (2 nM) of [3H]dihydrotestosterone [( 3H]DHT), approximately 50% of the total intracellular androgen receptor-steroid complexes were found in the nucleus. Within isolated nuclei from such cells, 28-49% of the specific androgen receptor binding was associated with the nuclear matrix. The antiandrogen cyproterone acetate inhibited DHT binding within the nuclear matrix. Cultured genital skin fibroblasts from two unrelated patients with receptor-positive complete androgen insensitivity (CAIS, AR+), had normal (approximately 50%) nuclear binding of DHT, and 35% and 45% of it was localized to the nuclear matrix. Genital skin fibroblasts from a patient with receptor-negative complete androgen insensitivity (CAIS, AR-) had no specific DHT binding in isolated nuclei or nuclear matrix. Scatchard analysis of specific DHT binding in the nuclear matrix isolated from cells of normal subjects after an in vitro exchange assay (0 C; 24 h) revealed the presence of saturable (maximum binding, approximately equal to 200 fmol/mg nuclear DNA), high affinity (Kd approximately equal to 1.0 nM) binding sites. By contrast, in the nuclear matrix isolated from cells of a patient with CAIS, AR+, the binding affinity for DHT was 3-fold lower (Kd approximately equal to 3.0 nM). When cytosolic androgen receptor-DHT complexes prepared from cells preincubated at 37 C for 1 h with [3H]DHT were incubated at 0 C for 1 h with isolated nuclei and nuclear matrix in the presence of 0.15 M KCl, 40-60% of specific nuclear binding was associated with the nuclear matrix. In these cell-free in vitro experiments, radiolabeled DHT-receptor complexes prepared from normal or mutant cells were mixed with isolated nuclei and nuclear matrix prepared from cells of normal subjects or patients with CAIS, AR+ or CAIS, AR-. Under these conditions, specific DHT binding in nuclei and nuclear matrix was quantitatively similar in the presence of a mutant (CAIS, AR+) receptor-steroid complex or in the presence of nuclei or nuclear matrix from the mutant cells (CAIS, AR- or AR+) when compared simultaneously with the same subcellular fractions prepared from the cells of normal subjects.(ABSTRACT TRUNCATED AT 400 WORDS)