Elsevier

Steroids

Volume 116, December 2016, Pages 20-27
Steroids

Modulation of the circadian clock by glucocorticoid receptor isoforms in the H295R cell line

https://doi.org/10.1016/j.steroids.2016.10.002Get rights and content

Highlights

  • A functional peripheral clock is present in H295R human adrenocortical cell line.

  • Glucocorticoids influence the expression of multiple clock genes.

  • GRß may modify the regulation of peripheral clock by glucocorticoids through REV-ERBα.

Abstract

Peripheral clocks are set by different nervous, hormonal and metabolic stimuli, and regulate the circadian expression of several genes. We investigated whether a peripheral clock could be induced in the human adrenocortical cell line H295R and whether glucocorticoid receptor isoforms (GRα and GRß) are involved in this clock system. After synchronization of cells with serum shock, the rhythmic oscillation of clock genes PER1, PER2, REV-ERBα, and ARNTL was confirmed. In addition, H295R cells even without serum shock showed rhythmic expression of PER1, PER2, CRY1 and ARNTL. Glucocorticoid treatment induced a rapid response of PER1, PER2 and CRY1 in a GRα-dependent manner. Continuous glucocorticoid stimulation after 6 h caused suppression of REV-ERBα. Administration of a GR antagonist, RU486, disrupted the circadian oscillation of clock genes and prevented the acute changes in PER1, PER2 and CRY1 levels. Overexpression of the GRß isoform alone did not alter the expression of the examined clock genes, but did prevent the GRα-related suppression of REV-ERBα. These alterations occurred independently from ACTH and CRH. Our data demonstrate that a peripheral clock system is present in a human adrenocortical cell line and that periodic oscillations of clock genes are influenced by glucocorticoids, mainly through GRα.

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).

References (53)

  • M. Surjit et al.

    Widespread negative response elements mediate direct repression by agonist-liganded glucocorticoid receptor

    Cell

    (2011)
  • R.H. Oakley et al.

    The dominant negative activity of the human glucocorticoid receptor beta isoform. Specificity and mechanisms of action

    J. Biol. Chem.

    (1999)
  • B.H. Miller et al.

    Circadian and CLOCK-controlled regulation of the mouse transcriptome and cell proliferation

    Proc. Natl. Acad. Sci. U.S.A.

    (2007)
  • K.-F. Storch et al.

    Extensive and divergent circadian gene expression in liver and heart

    Nature

    (2002)
  • P. Pezük et al.

    Glucocorticoids as entraining signals for peripheral circadian oscillators

    Endocrinology

    (2012)
  • M.E. Hughes et al.

    Harmonics of circadian gene transcription in mammals

    PLoS Genet.

    (2009)
  • J. Richards et al.

    Advances in understanding the peripheral circadian clocks

    FASEB J.

    (2012)
  • C.H. Ko et al.

    Molecular components of the mammalian circadian clock

    Hum. Mol. Genet.

    (2006)
  • J.a. Mohawk et al.

    Central and peripheral circadian clocks in mammals

    Annu. Rev. Neurosci.

    (2012)
  • a. Balsalobre et al.

    Resetting of circadian time in peripheral tissues by glucocorticoid signaling

    Science

    (2000)
  • T. Dickmeis

    Glucocorticoids and the circadian clock

    J. Endocrinol.

    (2009)
  • A.Y.-L. So et al.

    Glucocorticoid regulation of the circadian clock modulates glucose homeostasis

    Proc. Natl. Acad. Sci. U.S.A.

    (2009)
  • L.J. Lewis-Tuffin et al.

    Human glucocorticoid receptor beta binds RU-486 and is transcriptionally active

    Mol. Cell. Biol.

    (2007)
  • J. Fahrenkrug et al.

    Diurnal rhythmicity of the canonical clock genes Per1, Per2 and Bmal1 in the rat adrenal gland is unaltered after hypophysectomy

    J. Neuroendocrinol.

    (2008)
  • D.R. Lemos et al.

    Twenty-four-hour rhythmic gene expression in the rhesus macaque adrenal gland

    Mol. Endocrinol.

    (2006)
  • H. Oster et al.

    Transcriptional profiling in the adrenal gland reveals circadian regulation of hormone biosynthesis genes and nucleosome assembly genes

    J. Biol. Rhythms

    (2006)
  • Cited by (8)

    View all citing articles on Scopus
    View full text