Peripheral CLOCK Regulates Target-Tissue Glucocorticoid Receptor Transcriptional Activity in a Circadian Fashion in Man

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.

研究背景与研究目的
循环皮质醇在“主控”型生物钟CLOCK蛋白（circadian CLOCK）的调控下呈昼夜节律波动，而后者的外周“从属”型同源物则通过乙酰化作用，在局部糖皮质激素靶组织中调控糖皮质激素受体（GR）的转录活性。本研究旨在探讨人体内CLOCK介导的GR乙酰化对外周组织糖皮质激素敏感性的影响。
研究设计与研究对象
我们分别于上午8时和晚上8时，从10名健康受试者体内采集外周血单个核细胞（PBMCs），同时检测其中GR乙酰化水平以及GR、CLOCK相关基因和糖皮质激素应答基因的mRNA表达水平；此外，我们还采集了清晨的PBMCs并进行体外培养24小时，期间每6小时给予3小时的氢化可的松脉冲刺激。我们以EB病毒转化的淋巴细胞（EBVLs）作为非同步化对照。
研究结果
PBMCs中的GR乙酰化水平在清晨高于晚间，与循环皮质醇的波动呈反相相关。在非同步化的EBVLs中，所有检测过的已知糖皮质激素应答基因均能对氢化可的松产生预期的应答反应；但在体内的PBMCs中，部分此类基因并未呈现出预期的昼夜mRNA波动。而在缺乏内源性糖皮质激素的离体培养的天然同步化PBMCs中，这些基因的mRNA却呈现出依赖于CLOCK和GR乙酰化的节律振荡，这提示体内循环的皮质醇可能会抑制部分糖皮质激素应答基因中由昼夜节律GR乙酰化介导的效应。
研究结论
人体内外周CLOCK介导的GR昼夜节律乙酰化，可作为一种靶组织特异性、基因特异性的拮抗调控机制，对抗昼夜波动的皮质醇的作用：在清晨有效降低组织对糖皮质激素的敏感性，晚间则提升该敏感性。