Morphine potentiates glucocorticoid receptor translocation in neuronal cells

Abstract

Background: The hypothalamic-pituitary-adrenal (HPA) axis plays a central role in regulating signaling by glucocorticoid receptor which is expressed in almost all cells. Adrenocorticotropin hormone (ACTH) and β-endorphin both of which are derived through processing of pro-opiomelanocortin (POMC) pro-hormone are secreted from anterior pituitary under stressed conditions. ACTH released into circulation regulates the release of glucocorticoids from adrenal gland. Glucocorticoids cause profound suppression of functional activity of HPA axis as negative feedback control. The endogenous opioids acting primarily at mu opioid receptor inhibit activity of HPA axis and thus release ACTH and β-endorphin from anterior pituitary. Furthermore, there are enough reports to support that glucocorticoids regulate mu opioid receptor expression through GRE binding specially in mouse where it has already been shown that promoter region of mouse mu opioid receptor not delta or kappa opioid receptor contains a glucocorticoid-response element (GRE). The glucocorticoid receptor is a member of steroid-hormone receptor family of proteins. It binds to glucocorticoids with high affinity. In inactive state, the GR complexes with chaperones like heat shock proteins 70 (Hsp70) and 90 (Hsp90) and immunophilins and their co-chaperones making GR more accessible to ligand binding. After ligand binding the GR is activated and chaperones and co-chaperones are reshuffled with GR to be translocated to nucleus where the GR homodimerize and binds to GRE in promoter region. The resulting complex recruits either co-activator or co-repressor proteins that modify the structure of chromatin thereby facilitating or inhibiting assembly of the basal transcription machinery and the initiation of transcription by RNA polymerase II.

Aim: Acute and chronic morphine treatments have been shown to result in marked induction of Hsp70 messenger RNA expression. At the same time Hsp70 is also an integral part of GR assembly in inactive and active state. We are therefore hypothesizing that mu opioid receptor crosstalk with glucocorticoid receptor, and this interaction is in part, mediated by Hsp70 acting as adaptor protein.

Method: Murine N2A cells were either stably transfected with HA-tagged mu opioid receptor and/or HSP70. These cells were treated with morphine sulphate (1uM) for 5 hours followed by treatment with different concentrations of corticosterone (GC) for 30 minutes. We confirmed their crosstalk by immunoprecipitation and co-immunoprecipitation experiments. Further, nuclear translocation of glucocorticoid receptor was monitored in immunoblots.

Results and Discussion: Morphine treatment resulted in increased expression of Hsp70. Pull-down assays showed the interaction between mu opioid receptor and glucocorticoid receptor. These two proteins also interacted with Hsp70; a chaperone protein known to interact with glucocorticoid receptor. Morphine treatment potentiated glucocorticoid receptor translocation to the nucleus which was further potentiated by Hsp70 overexpression. These results confirm the crosstalk between MOR and GR. For future studies, we would study this interaction in immune cells like J774 (alveolar macrophages) and CRL2019 cells (peritoneal macrophages).