Dexamethasone does not exert direct intracellular feedback on steroidogenesis in human adrenal NCI-H295A cells

Abstract

Experimental therapy of fetuses affected with congenital adrenal hyperplasia (CAH) has been reported by administering dexamethasone (Dex) to pregnant women at risk for carrying a CAH fetus. Such prenatal therapy can almost wholly eliminate virilization of the external genitalia of affected female fetuses, but only when treatment is started before 9 weeks of gestation. As it is not known whether the hypothalamic-pituitary-adrenal axis is functional at this time, and as the minimal effective doses of Dex are substantially supraphysiologic for the fetus, the mechanism of action of prenatal Dex treatment has been unclear.To assess the possibility that Dex might act directly on the adrenal, we used human adrenocortical NCI-H295A cells, an established model of the human fetal adrenal. Short term (6 h) incubation of these cells with Dex decreased synthesis of 11-deoxycortisol and 17 -hydroxyprogesterone and increased synthesis of deoxycorticosterone (DOC), but only at very high concentrations of Dex (d10 µM) that affect P450c17 (17 -hydroxylase/17,20 lyase) as a competitive inhibitor; no effects were seen at lower concentrations corresponding to those used in prenatal treatment. When NCI-H295A cells were treated with up to 100 µM Dex for 72 h, dot-blot analysis showed no changes in the abundance of the mRNAs for P450scc (cholesterol side-chain cleavage enzyme), P450c17, or 3 -hydroxysteroid dehydrogenase II (3 HSDII). When NCI-H295A cells were transfected with promoter/reporter constructs for the human genes for P450scc and P450c17, 24-h treatment with Dex at doses up to 100 µM had no effect on the transcription of these constructs. Because the regulation of steroidogenic processes in NCI-H295A cells closely resembles such regulation in the adrenal, we suggest that Dex may not act by an intra-adrenal mechanism in the experimental prenatal treatment of CAH.

Colon cancer cells produce immunoregulatory glucocorticoids

“…14 C-labeled progesterone ([4-14 C]-progesterone, 55 mCi/mmol, American Radiolabeled Chemicals Inc., St Louis, MO, USA) was added per well. Cell supernatant was harvested at various time points and steroids were extracted as described elsewhere (Dardis and Miller, 2003). Samples were then separated by thin-thin-layer chromatography using methylene chloride:methanole:H 2 O (200:30:1) as solvent system.…”

Effect of glucocorticoids pretreatment on steroidogenic capacity of adrenocortical cells isolated from Meishan piglets

“…Most of these studies showed that acute and/or chronic exposure of adrenal cells to exogenous glucocorticoids reduces ACTH-induced steroidogenesis (Carsia and Malamed 1983;Tim et al 1999). However, other studies suggest a positive effect of glucocorticoids on the steroid output of adrenocortical cells (Kahri et al 1979;Dardis and Miller 2003). It has been proposed that glucocorticoids may decrease the binding of ACTH to its receptor, thus inhibiting ACTH-induced cAMP production (Latner et al 1977), while it was also reported that exogenous steroids might enhance steroid hydroxylase activity (Saito et al 1979).…”

Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3β-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

“…In contrast to the parental H295 cells, both A and R cells grow as a tight monolayer. Similar to H295 cells, A and R cells express all genes involved in adrenal steroidogenesis (Staels et al 1993, Rodriguez et al 1997, Dardis & Miller 2003. However, all investigators who study adrenal steroidogenesis use either A or R cells and compare their research data directly assuming that the two cell lines are identical.…”