Phosphorylation of leukocyte glucocorticoid receptor in patients with current episode of major depressive disorder Iva Simic a , Nadja P. Maric b, c , Milos Mitic a , Ivan Soldatovic c , Zorana Pavlovic b , Marina Mihaljevic b , Sanja Andric b , Marija B. Radojcic a , Miroslav Adzic a, ⁎ a Laboratory of Molecular Biology and Endocrinology, VINCA Institute of Nuclear Sciences, University of Belgrade, P.O. Box-522-MBE090, 11001 Belgrade, Serbia b Clinic for Psychiatry, Clinical Centre of Serbia, Pasterova 2, 11000 Belgrade, Serbia c Faculty of Medicine, University of Belgrade, Dr Subotica 8, 11000 Belgrade, Serbia abstract article info Article history: Received 6 September 2012 Received in revised form 11 October 2012 Accepted 26 October 2012 Available online 1 November 2012 Keywords: Glucocorticoid receptor Major depressive disorder Phosphorylation The impaired glucocorticoid receptor (GR) signaling has long been considered one of the cornerstones in understanding the pathophysiology of depression. Since the phosphorylation of GR is very important for GR function, in this study we investigated whether GR phosphorylation at serine 211 (pGR-S211) and serine 226 (pGR-S226) is altered in patients with current episode of major depressive disorder (MDD). Particularly, in 30 MDD patients and 35 controls we assessed the levels of nuclear total GR (tGR), pGR-S211 and pGR-S226 in peripheral blood mononuclear cells (PBMC) using Western blot technique, along with plasma cortisol con- centrations from the same blood samples. Our results demonstrated increased phosphorylation of GR at S226 (p b 0.001) and, to a less extent, at S211 (p b 0.05) in MDD patients compared to controls. Consequently, the pGR-S211/pGR-S226 ratio was decreased (p b 0.05) implying reduced transcriptional activity of GR in MDD patients. MDD subjects had higher cortisol levels than controls and cortisol concentrations were positively correlated with PBMC pGR-S226 levels from the same blood samples. There was no difference in the levels of tGR between MDD and control subjects. The study showed that altered phosphorylation of GR could con- tribute to impaired GR function related to the pathophysiology of depression. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Altered hypothalamic–pituitary–adrenal (HPA) axis activity has been reliably observed in patients with mood disorders, while gluco- corticoids have long been considered among key players in their pathogenesis (Anacker et al., 2011; Holsboer, 2000; Pariante and Lightman, 2008). Thus, in order to better understand the underlying pathophysiology of depressive disorders, the research has been focused on glucocorticoid effects mediated by intracellular receptors including, most notably, the glucocorticoid receptor (GR). A list of studies has considered the possibility that the number of GRs, differ- ent GR isoforms or GR chaperones are altered in depressed patients (Binder, 2009; Holsboer, 2000; Matsubara et al., 2006; Pariante and Miller, 2001; Yehuda et al., 1993), while more recent research focuses on GR-stimulated changes in gene expression (Menke et al., 2012). Phosphorylation of GR is a very important way of regulating GR function. It affects GR stability, nuclear-cytoplasmic shuttling and its interactions with other transcriptional factors ultimately leading to different regulations of GR-responsive genes (Galliher-Beckley and Cidlowski, 2009; Ismaili and Garabedian, 2004; Itoh et al., 2002). Human GR can be phosphorylated at serine 211 (pGR-S211) and serine 226 (pGR-S226) by diverse kinases such as cyclin-dependent kinases (CDKs) and mitogen-activated protein kinases (MAPKs) (Krstic et al., 1997; Miller et al., 2005; Rogatsky et al., 1998). The GR phosphorylation at S211 promotes GR translocation to the nucleus and enhances its tran- scriptional activity (Chen et al., 2008; Wang et al., 2002), while GR phosphorylation at S226 inhibits its transcriptional activity and pro- motes GR nuclear export (Chen et al., 2008; Itoh et al., 2002). The ratio of pGR-S211/pGR-S226 may be viewed as an important parameter which determines GR transcriptional activity and expression of GR-responsive genes (Blind and Garabedian, 2008; Chen et al., 2008). In our previous experiments it was demonstrated that chronically stressed animals (Wistar rats) which exhibited anxiety-like and depressive-like behavior, had also prominent alterations in rodent homologues of human pGR-S211 and pGR-S226 in nuclear extracts of hippocampus and prefrontal cortex (Adzic et al., 2009a; Djordjevic et al., 2012). These changes in the GR phosphorylation pattern coincided with altered mitochondrial energy production, cell redox imbalance, compromised cellular plasticity and onset of Progress in Neuro-Psychopharmacology & Biological Psychiatry 40 (2013) 281–285 Abbreviations: GR, glucocorticoid receptor; pGR-S211, GR phosphorylated at serine 211; pGR-S226, GR phosphorylated at serine 226; S211, serine 211; S226, serine 226; MDD, major depressive disorder; PBMC, peripheral blood mononuclear cells; tGR, total glucocorticoid receptor; IRS, internal reference sample. ⁎ Corresponding author at: Laboratory of Molecular Biology and Endocrinology, VINCA Institute of Nuclear Sciences, P.O. Box-522-MBE090, 11001 Belgrade, Serbia. Tel.: +381 11 3408304; fax: +381 11 2455561. E-mail address: miraz@vinca.rs (M. Adzic). 0278-5846/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2012.10.021 Contents lists available at SciVerse ScienceDirect Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp