Objectives: The main objectives of this study were: a) to determine whether male and female hormones regulate the expression of RORA in a neuronal cell line, SH-SY5Y; 2) to examine whether or not RORA transcriptionally regulates aromatase, an enzyme that converts testosterone to estrogen; and 3) to investigate the relationship between RORA and aromatase expression in the frontal cortex of autistic vs. sex- and age-matched nonautistic controls.
Methods: Neuroblastoma cells SH-SY5Y were cultured and treated with 17β-estradiol or dihydrotestosterone (DHT). Dose-response and time-course studies of the sex hormones were conducted using RT-qPCR analyses. Chromatin immunoprecipitation (ChIP)-PCR experiments were used to demonstrate that RORA is a transcriptional target of androgen receptor (AR) and estrogen receptor alpha (ERα), and also that aromatase is a transcriptional target of RORA. To further validate that RORA can modulate aromatase expression, the cells were transfected with a RORA expression plasmid, and aromatase expression was then determined by RT-qPCR analyses. Confocal immunofluoresecence analysis was conducted using a tissue array containing postmortem brain tissues (BA9 region) from autistic and sex- and age-matched control subjects to determine the expression and relationship of RORA and aromatase protein levels in the brain.
Results: Our findings reveal that male and female hormones differentially regulate the expression of RORA. In addition, we demonstrate that RORA transcriptionally regulates aromatase (CYP19A1), an enzyme that converts testosterone to estrogen. Finally, we show that aromatase protein is reduced and strongly correlated with the level of RORA protein in brain tissues from autistic and control subjects.
Conclusions: These results collectively indicate that RORA has the potential to be under both negative and positive feedback regulation by male and female hormones, respectively, through one of its target genes, aromatase. These findings not only offer a molecular explanation for the elevated testosterone levels found in some individuals with autism, but also suggest a mechanism for introducing sex bias in autism.