Autoregulation of androgen receptor by androgen in mouse brain: Characterizing the mechanism of androgen action

Androgen receptor (AR), a member of the nuclear receptor superfamily, is a critical mediator of androgenic signaling. Deficiencies in either receptor concentration and/or functional domains of the receptor lead to androgen insensitivity syndrome, indicating that a normal level and function of AR protein is critical. Previous data suggested that ligand induced up-regulation of cellular AR content represents a critical step in androgen action. To further test this hypothesis, several factors including sex, androgen dosage, and type of ligand were assessed for their effects on the expression of AR in mouse brain through quantitative ICC and western blot. To explore the relationship between androgen induced changes in AR and AR mRNA, a ribonuclease protection assay (RPA), combined with western blot, was employed to measure the time-course changes of these molecules after T treatment. It was demonstrated that gonadectomy (GDX), which removes virtually all endogenous androgens, caused a significant loss of AR. Replacement with testosterone (T) or dihydrotestosterone (DHT) restored AR back to or above that seen in intact animals. The extent of increase in AR correlated linearly to the androgen dose. This regulatory pattern was seen in both male and females. Moreover, the extent of AR up-regulation by different ligands appeared to reflect their relative hormonal potencies. DHT, which is a more potent androgen than T based on its higher affinity for AR, had a longer effect on receptor augmentation compared to T. A pure AR antagonist, flutamide, showed little effect on AR concentration when administered alone to GDX mice. It inhibited agonist induced AR augmentation when injected concomitantly with T or DHT at a dose ratio of 100 to 1. In these experiments, a positive correlation between physiological function and the ability of a ligand to upregulate AR was demonstrated. Interestingly, both T and flutamide significantly increased nuclear AR signal, suggesting that the requirement of ligand binding for nuclear translocation of AR is not agonist specific. Finally, down-regulation of steady state AR mRNA content by T was observed over a 24-hr period by RPA. There apparently is an absence of a systematic, positive relationship between changes in AR mRNA and AR protein induced by T, indicating that androgen induced up-regulation of AR is independent of changes in AR mRNA The current results support the concept that agonists, augment AR level by increasing AR stability.