Study of the Signaling Function of Sulfiredoxin and Peroxiredoxin III in Isolated Adrenal Gland: Unsuitability of Clonal and Primary Adrenocortical Cells

Abstract

Members of the peroxiredoxin (Prx) family of antioxidant enzymes are inactivated via hyperoxidation of the active site cysteine by the substrate H2O2 and are reactivated via an ATP-consuming process catalyzed by sulfiredoxin (Srx). PrxIII is reversibly inactivated by H2O2 produced by cytochrome P450 11B1 (CYP11B1) in mitochondria during corticosterone synthesis in the adrenal gland of mice injected with adrenocorticotropic hormone (ACTH). Inactivation of PrxIII triggers a sequence of events including accumulation of H2O2, activation of p38 mitogen-activated kinase (MAPK), inhibition of cholesterol transfer, and suppression of corticosterone synthesis. Srx expression is significantly induced by ACTH injection. The coupling of CYP11B1 activity to PrxIII inactivation and Srx induction provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis. Furthermore, the PrxIII-Srx regulatory pathway is critical for the circadian rhythm of corticosterone production. Although adrenocortical tumor cell lines such as Y-1 and H295R have been used extensively for studying the mechanism of steroidogenesis, those clonal cells were found to be unsuitable as an in vitro model for redox signaling because the amount of Srx in the cell lines is much higher than that in mouse adrenal gland and not affected by ACTH stimulation. Furthermore, the levels of PrxIII in the clonal cells are greatly reduced compared to that in the adrenal gland, and ACTH does not induce PrxIII hyperoxidation in the clonal cells. Primary adrenocortical cells isolated from the mouse adrenal gland were also found to be an invalid model because Srx levels are increased, along with decreased levels of hyperoxidized PrxIII, soon after isolation of these cells. Organ culture system is, however, appropriate for studying the PrxIII-Srx regulatory function as the levels of hyperoxidized PrxIII and Srx in the adrenal glands maintained overnight in culture medium are not changed.