Abstract

Context and ObjectiveCirculating cortisol fluctuates diurnally under the control of the “master” circadian CLOCK, while the peripheral “slave” counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR) at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans.Design and ParticipantsWe examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs) obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs) as non-synchronized controls.ResultsGR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo.ConclusionsPeripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night.

Highlights

  • Human beings live under the strong influence of light/dark cycles associated with the day/night changes created by the 24hour rotation of the earth [1]

  • All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized Epstein-Barr virus (EBV)-transformed lymphocytes (EBVLs), some of these genes did not show the expected diurnal mRNA fluctuations in peripheral blood mononuclear cells (PBMCs) in vivo

  • To further examine the physiologic interaction of the circadian CLOCK system and the HPA axis at peripheral glucocorticoid target tissues in humans, we performed an in vivo clinical study in which we examined the acetylation of the glucocorticoid receptor (GR), as well as the mRNA expression of CLOCK-related and glucocorticoidresponsive genes employing peripheral blood mononuclear cells (PBMCs) from healthy adult subjects

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Summary

Introduction

Human beings live under the strong influence of light/dark cycles associated with the day/night changes created by the 24hour rotation of the earth [1]. The SCN acts as a ‘‘master’’ CLOCK under the strong influence of light/dark input from the eyes, whereas the peripheral CLOCK behaves as a ‘‘slave’’, subjugated by the former through as yet unclear mechanisms [4] Both master and slave CLOCKs share almost the same transcriptional regulatory machinery with coordinated activation/inactivation of a set of transcription factors, including the ‘‘circadian locomotor output cycle kaput’’ (Clock), its heterodimer partner ‘‘brainmuscle-arnt-like protein 1’’ (Bmal1) and other essential negative regulators, such as the ‘‘Periods’’ (Pers), ‘‘Cryptochromes’’ (Crys), and the nuclear hormone receptors REV-erbs and retinoic acid receptor-related orphan receptors (RORs) [1]. These transcription factors create a negative feedback transcriptional loop through mutual transcriptional activation and repression that maintains an approximately 24-hour oscillation of their gene expression [1]

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