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Pauliina Utriainen, Jianqi Liu, Tiina Kuulasmaa and Raimo Voutilainen

Activin affects adrenocortical steroidogenesis and increases apoptosis, while follistatin (FS) acts as an activin antagonist by binding to activin, preventing attachment to its receptors. The regulation of FS expression in the adrenal cortex is poorly understood. Adrenocortical tumors often display aberrant methylation. In the present study, we investigated the effect of DNA methylation on FS mRNA expression and peptide secretion in adrenocortical cells. We treated human NCI-H295R adrenocortical cells with the methylation inhibitor 5-Aza-2′deoxycytidine (Azad; 0.1–100 μM for 1, 4 or 7 days) and measured FS mRNA expression by Northern blot and quantitative real time RT-PCR analyses as well as FS secretion by specific ELISA. Methylation-specific PCR showed decreased methylation in the FS promoter region after Azad treatment. A significant (P < 0.05) time- and dose-dependent increase in FS mRNA expression (up to 4.6-fold) and peptide secretion (up to 17.1-fold) was detected after Azad treatment. We conclude that FS gene expression and peptide secretion in NCI-H295R adrenocortical cells are regulated by DNA methylation. Thus, variable methylation in different adrenocortical tumors may influence activin bioactivity and its consequences in steroidogenesis and cell proliferation/apoptosis.

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Irina V Mikhaylova, Tiina Jääskeläinen, Jarmo Jääskeläinen, Jorma J Palvimo and Raimo Voutilainen

Leukemia inhibitory factor (LIF) is a multiple function cytokine regulating the hypothalamic–pituitary–adrenal axis at the pituitary level. LIF and its receptor are expressed in the adrenal glands, suggesting their potential regulatory role also at the adrenal level. Our aim was to clarify the effects of LIF on adrenal steroidogenesis using cell culture conditions. NCI-H295R human adrenocortical cells were treated with LIF (0.01–100 ng/ml) for 3–48 h with or without 8-bromo-cAMP (8-Br-cAMP; 1 mM). LIF treatment augmented cortisol, dehydroepiandrosterone (DHEA), DHEA sulfate, androstenedione, and aldosterone production (up to 224, 211, 149, 229, and 170% of control respectively, P<0.05 for all). It increased basal steroidogenic acute regulatory protein (STAR) and 17α-hydroxylase/17,20-lyase (CYP17A1) mRNAs (up to 142 and 170% of control respectively, P<0.05) and the respective proteins, but decreased 3β-hydroxysteroid dehydrogenase type 2 (HSD3B2) mRNA (down to 72% of control, P<0.05), and protein. LIF also increased 8-Br-cAMP-induced cortisol and DHEA production and STAR mRNA accumulation, while it attenuated 8-Br-cAMP-induced HSD3B2 expression and androstenedione production. It had an additive effect on tumour necrosis factor-induced cortisol production. LIF had no effect on apoptosis, but it increased slightly the number of metabolically active cells (up to 120% of control, P<0.05). These findings indicate that LIF is a potential physiological and/or pathophysiological regulator of steroidogenesis at the adrenal level.