VIJAYAN, M.M.; SATHIYAA, R.; University of Waterloo; University of Waterloo: Autoregulation of glucocorticoid receptor by cortisol in rainbow trout hepatocytes

Glucocorticoids play an important role in the stress response and glucocorticoid receptor (GR) regulation may be a key process in the physiological response to regain homeostasis. We used primary cultures of rainbow trout hepatocytes and a physiological dose of cortisol (100 ng ml^-1), mimicking stressed levels in salmonid fish, to address the impact of glucocorticoid stimulation on GR mRNA abundance and protein content. Cortisol significantly elevated GR mRNA content over a 24 h period and this increase was abolished by actinomycin D suggesting transcriptional control of GR. However, the GR protein content was significantly lower in the cortisol treated group leading us to hypothesize that this lower GR protein content may be regulating GR mRNA abundance. Indeed, treatment of hepatocytes with MG-132, a proteasomal inhibitor that prevented GR protein degradation by cortisol, abolished cortisol-mediated GR mRNA upregulation. Geldanamycin, an hsp90- specific inhibitor, abolished the GR mRNA increase evident with cortisol suggesting a role for hsp90 in this GR autoregulation. However, geldanamycin did not modify cortisol-induced increases in abundance of mRNA for PEPCK, a glucocorticoid-responsive gene, or hepatocyte glucose release reflecting a direct impact of hsp90 on GR transcription and not at the level of GR protein regulation. Together, our results, using a physiologically relevant model system, suggest a negative feedback loop for GR gene regulation in trout hepatocytes. We also show that hsp90 and the proteasome may be directly involved in this GR autoregulation by cortisol.