Endocrine Abstracts

Background: Peripheral clocks are set by different nervous, hormonal and metabolic stimuli and regulate the circadian expression of several genes. It has been demonstrated that circadian oscillation of gene expression can be detected in various cell lines in vitro.

Aim: To explore whether a peripheral clock could be induced in human adrenocortical cell line H295R and what are the effects of glucocorticoids on this clock system.

Methods: H295R human adrenocortical cell line was studied. For serum shock experiments cells were serum starved for 24 h and incubated with 30% Nu serum for 2 h then returned to normal medium with either vehicle or 100 nmol dexamethasone (Dex). For Dex experiments cells were serum starved for 24 h, maintained in serum free medium and treated with vehicle or 100 nmol Dex with or without 1 μmol RU486. Cells were harvested at the indicated time points. All experiments were carried out in triplicate. RT-PCR was carried out using predesigned TaqMan gene expression assays. PER1, PER2, CRY1, ARNTL, NR1D1, NR3C1, StAR, POMC, CRH, and actin expressions were measured by qRT-PCR on 7500 Fast Real-Time PCR System.

Results: After synchronisation of cells the rhythmic oscillation of clock genes PER1, PER2, NR1D1, and ARNTL was observed. Glucocorticoid treatment induced a rapid respond of PER1 in a glucocorticoid receptor (GR)-dependent manner. Continuous glucocorticoid stimulation caused elevation of PER1 and altered its rhythm. Glucocorticoid treatment induced the expression of PER2 and delayed its phase, increased expression of CRY1 and, after 6 h, it suppressed expression of NR1D1. Administration of a GR antagonist, RU486 disrupted the circadian oscillation of clock genes and prevented the acute changes in PER1, PER2 and CRY1 levels. These alterations occurred independently from ACTH or CRH.

Conclusions: Our data demonstrated that a peripheral clock system is present in human adrenocortical cell line and periodic oscillation of clock genes are influenced by glucocorticoids.

Disclosure: The authors received financial support from Hungarian Scientific Research Fund (OTKA PD-100648) and National Development Agency (KTIA-AIK-10_2012-0010).