Modulation of the circadian clock by glucocorticoid receptor isoforms in the H295R cell line
Introduction
Circadian timing of gene expression is an important and evolutionary-conserved regulatory mechanism maintaining physiological processes [1], [2] and ensuring the physiological adaptation of the organism to the day/night cycle. The SCN in the hypothalamus is the main circadian synchronizer of the organism [3], [4], [5]. Recently it has been shown that beside the central clock in the SCN, almost every tissue in the body possesses a peripheral circadian clock machinery [6]. Peripheral clocks drive the circadian expression of several genes in a tissue-specific manner [2]. The molecular circadian clock machinery consists of two interacting feed-back loops. In the primary loop, positive transcription regulators ARNTL (BMAL1) and CLOCK transcription factors heterodimerize and stimulate the transcription of period (PER1, PER2, PER3) and cryptochrome (CRY1, CRY2) genes. However, PER and CRY proteins can heterodimerize and, by inhibiting the ARNTL/CLOCK complex, repress their own transcription [7]. The ARNTL/CLOCK complex activates the transcription of nuclear receptors RORα (NR1F1) and REV-ERBα (NR1D1), forming the accessory loop of the molecular clock machinery. ARNTL is positively regulated by RORα and negatively regulated by REV-ERBα [7]. Post-transcriptional mechanisms have also been demonstrated in the maintenance of the approximately 24 h periodicity of the expression of clock genes [7].
Peripheral clocks are set to external time by different regulatory (neural, hormonal, temperature, metabolic control) pathways [8]. Among these factors glucocorticoids are highly potent agents in the synchronization of peripheral clocks. They are able to modify and reset the circadian clock in many tissues and even in immortalized cell lines [4], [9], [10], [11]. However, the underlying mechanism of glucocorticoids in the entrainment of peripheral clocks is not understood. Glucocorticoids exert their effect through the GRα isoform. After ligand binding, GRα forms homodimers and regulates transcription directly by binding glucocorticoid responsive elements (GRE) or indirectly by the modulation of other transcription factors. The GRß isoform exerts a dominant negative activity on GRα function. Recently it was shown that GRß may also have a GRα-independent transcriptional activity in various cell lines [12], [13], [14]. The adrenal gland, through the secretion of cortisol, has an important role in the regulation of peripheral circadian oscillators [4]. Nevertheless, animal experiments demonstrated a functional circadian clock in the adrenal gland [15], [16], [17]. The adrenal clock seems to be essential for the daily rhythmic generation of glucocorticoids [18]. In peripheral Cushing’s disease, the daily pattern of cortisol secretion is disturbed and steroid production is not under the control of the hypothalamus-hypophysis-adrenal (HPA) axis [19], [20]. In adrenal neoplasms, the lack of higher control may give rise to cell-autonomous regulatory mechanisms [20]. In cortisol-producing adrenal adenomas, the expression of both GRα and GRß are increased [19], and glucocorticoid receptor (GR) was recently shown to be involved in the autocrine regulatory feedback of steroid production in adrenocortical cells. Thus, glucocorticoid feedback on the adrenal clock would imply a particular connection between the two systems. Therefore, we aimed to test whether glucocorticoids could regulate the adrenal peripheral clock in the human adrenocortical cell line H295R, and we aimed to identify the possible intervention points of GRα and GRß isoforms in this regulation.
Section snippets
Cell culture
The H295R cell line was grown in Dulbecco’s modified Eagle’s medium and Ham’s F12 Nutrient Mixture (1:1) supplemented with 15 mM HEPES, 6,25 μg/ml insulin, 6,25 μg/ml transferrin, 6,25 ng/ml selenium, 1,25 mg/ml bovine serum albumin, 5,35 μg/ml linoleic acid and 2,5% Nu-Serum. Cells were cultured in a humidified incubator infused with 5% CO2 at 37 °C. All compounds were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Circadian experiments
H295R cells were plated at a density of 106 cells/well on 6-well tissue culture
Peripheral clock is functional in H295R adrenocortical cells
After synchronization of cells with serum shock, we detected the rhythmic expression of 4 clock genes: PER1, PER2, REV-ERBα and ARNTL by cosinor analysis. CRY1 levels became elevated upon serum shock, but did not show any rhythmic oscillation (Fig. 1). The expression pattern of clock genes was consistent with a regulatory feedback mechanism: PER1 and PER2 oscillated in the same phase, whereas REV-ERBα and ARNTL showed an anti-phase pattern.
Transcriptional regulation of total GR by glucocorticoids in H295R cells
We evaluated the expression pattern of total GR in the
Discussion
In our study we first aimed to test whether the human adrenocortical cell line H295R possesses a functional peripheral circadian clock. Serum shock has been previously reported to induce clock genes in a mammalian fibroblast cell line [23]. After serum shock treatment our data confirmed that in the H295R cell line, circadian expression of clock genes can be induced and that the expression pattern of different clock genes was consistent with a regulatory feedback mechanism, indicating an intact
Acknowledgments
The authors receive financial support from Hungarian Academy of Sciences “Lendulet 2013” Grant (AP), Bionics Innovation Center, Budapest, Hungary (AP, IL) and National Development Agency, Hungary (KTIA-AIK-10_2012-0010).
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