Abstract
Abstract: :
Purpose: Retinoic acid (RA) down–regulates androgen receptors (AR) in lacrimal gland and inhibits growth of lacrimal acinar cells in culture. If RA decreases AR levels, then treatment of cells with RA might inhibit downstream genomic responses to androgens. This study investigated whether RA would inhibit the androgen response in a lacrimal cell line expressing an androgen response element (ARE) linked to a luciferase (Luc) reporter gene. Methods: Transformed rabbit lacrimal acinar cells were transfected with TAT–E1BTATA–Luc which has a glucocorticoid response element (GRE) that also acts as an ARE, and also with pCH110 which contains the ß–galactosidase (ß–gal) gene. Cells were maintained in glucocorticoid–free, OptiMEM medium in plates coated with Matrigel. In initial experiments cells were exposed to dexamethasone (Dex) that was present in the HepatoSTIM medium used to dilute the Matrigel. A second series of experiments was done using Matrigel applied to the plates in OptiMEM. Cells were exposed to 10–6 M RA, in ethanol vehicle, for 24 hr and then treated with 10–9–10–6M dihydrotestosterone (DHT) in the presence of RA for 48 hr. Control cells were transfected and exposed to ethanol vehicle alone. Luc and ß–gal activity and total protein were measured. Results:ß–gal activity was reduced 2X compared to control in the presence of RA, so Luc activity was normalized to total protein. The presence of Dex caused an 8X induction of Luc activity over baseline levels, while DHT+Dex caused an 11X induction of Luc. There was no dose–response relationship between DHT and Luc activity. RA caused a 1.7X reduction in Dex–stimulated Luc activity and a 4.1X reduction when Dex and DHT were both present. In contrast, when Dex was absent DHT did not induce Luc, but RA itself caused a 1.85X induction of Luc above baseline, in the presence or absence of DHT. Conclusions: The construct used in this study was highly sensitive to glucocorticoid since the low level of Dex (concentration unknown) present when Matrigel was applied in HepatoSTIM strongly induced Luc. DHT and Dex appear to interact since DHT potentiated the response to Dex, but DHT did not induce Luc by itself. This may be due to the very low levels of AR in this cell line. The induction of Luc activity by RA alone, as well as inhibition of ß–gal, was unexpected. Retinoid signaling is mediated by a complex system of nuclear receptors. It is unknown whether retinoid receptors interact directly with the constructs used in this study, but Luc and ß–gal may be indirectly influenced by other gene products whose expression is controlled by RA.
Keywords: lacrimal gland • cornea: tears/tear film/dry eye • retinoids/retinoid binding proteins