Methylation of glucocorticoid receptor gene promoter modulates morphine dependence and accompanied hypothalamus–pituitary–adrenal axis dysfunction

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Opioid dependence is a chronic relapsing brain disorder characterized by sustained drug craving and compulsive drug taking (Edwards and Koob, 2010). Stress neurocircuitry mediates the development and maintenance of opioid dependence (Koob and Kreek, 2007; Chaijale et al., 2013). Specifically, opioid dependence is accompanied by complex and long‐lasting changes in hypothalamus–pituitary–adrenal (HPA) axis function.
Rats exhibit hypersecretion of corticosterone in response to restraint stress and reduced sensitivity to steroid negative feedback in the short term following morphine dependence (Houshyar et al., 2001a). Morphine withdrawal also stimulated the secretion of corticosterone (Laorden et al., 2002; Cleck and Blendy, 2008). Additionally, clinical studies revealed an aberrant activity of the HPA axis in opiate addicts, and its normalization in these patients predicted an efficient clinical therapy (Zhang et al., 2008). Recent studies demonstrated that morphine‐induced dysfunction of the HPA axis might be involved in the transition from occasional drug use to dependence (Koob and Kreek, 2007; Koob et al., 2014) and stress‐triggered drug seeking and relapse (Yang et al., 2004; Nunez et al., 2009; Navarro‐Zaragoza et al., 2015). However, the molecular mechanism underlying morphine‐induced HPA dysfunction, and its role in morphine dependence, remains unclear.
Glucocorticoid receptor (GR) is the prime mediator of glucocorticoid action in the brain (Herman and Spencer, 1998). Increasing evidence suggests that hippocampal GR plays a critical role in modulating the neuroadaptation of brain stress neurocircuitry to stressors (Zhang et al., 2013; van der Knaap et al., 2015). For instance, hippocampal GR was found to mediate the negative feedback inhibition of the HPA axis (Jacobson and Sapolsky, 1991) and the persistent alteration of HPA axis function induced by multiple environmental factors (Feldman and Weidenfeld, 1999; Witzmann et al., 2012; Zhang et al., 2013). Moreover, GR is a major molecular substrate of the properties of addictive drugs (De Vries et al., 1996; Ambroggi et al., 2009; Barik et al., 2010). Blockade of GRs reduces opiate‐related signaling molecule activation (Navarro‐Zaragoza et al., 2015) and suppresses morphine‐induced hyperlocomotor and dopamine release in the nucleus accumbens (Marinelli et al., 1998). Paradoxically, it is also reported that suppression of GR expression by a transgenic approach enhances morphine‐induced hyperlocomotor and mesolimbic dopamine release (Spanagel et al., 1996; Sillaber et al., 1998). All these findings suggested a complex role of GR signaling in modulating HPA axis function and morphine dependence. However, the exact role of hippocampal GR in morphine dependence, especially in the dysfunction of the HPA axis following opioid dependence, and the underlying mechanism, remains unclear.
The expression of GR was modulated at multiple levels, including the transcriptional, the posttranscriptional, and the posttranslational levels (Vandevyver et al., 2014). Recent studies found that epigenetic modification of hippocampal GR promoter and subsequent expression alteration modulate HPA function and contribute to multiple mental disorders (Turecki and Meaney, 2016). For instance, hypermethylation of the GR gene promoter 17 variant induced by early‐life social stresses was found to downregulate hippocampal GR expression and lead to HPA axis dysregulation (Daniels et al., 2009; Vukojevic et al., 2014). DNA methylation is also an important mechanism underlying chronic morphine–induced persistent neuroplasticity (Nestler, 2014). However, the effects of GR promoter methylation on morphine‐induced alteration in GR expression, HPA axis function, and morphine dependence are still unclear.
In the current study, we first evaluate the effects of the chronic morphine exposure on hippocampal GR expression and its promoter methylation. Next, we manipulated the methylation of hippocampal GR promoter with 5′‐aza‐2′‐deoxycytidine (5‐aza), an inhibitor of DNA methyltransferases (DNMTs), to examine the effects of GR promoter methylation on morphine‐induced HPA axis hyperreactivity and the expression of somatic withdrawal symptoms in rats, as well as the underlying mechanism.
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