Recent advances in the molecular mechanisms determining tissue sensitivity to glucocorticoids: novel mutations, circadian rhythm and ligand-induced repression of the human glucocorticoid receptor

In humans, glucocorticoids are synthesized by the adrenal cortex and released following activation of the hypothalamic-pituitary-adrenal (HPA) axis, and play an important role in the maintenance of resting and stress-related homeostasis. Named for their effects on glucose metabolism, glucocorticoids are involved in almost every cellular, molecular and physiologic network of the organism, and regulate a broad spectrum of physiologic functions essential for life, including growth, reproduction, cognition, behavior, cell proliferation and survival, as well as immune, central nervous system (CNS) and cardiovascular functions [1‐3]. Given their powerful anti-inflammatory and immunosuppressive actions, synthetic glucocorticoids represent one of the most widely used therapeutic compounds employed in the treatment of acute and chronic inflammatory/autoimmune and lymphoproliferative disorders [1, 3]. However, chronic exposure to glucocorticoids in patients with such disorders leads to multiple adverse effects. Sometimes glucocorticoid resistance of the affected organ or tissue may develop, representing a major challenge for the treatment of these conditions [4].

At the cellular level, the actions of glucocorticoids are mediated by the human glucocorticoid receptor (hGR, NR3C1), which belongs to the steroid/thyroid/retinoic acid nuclear receptor superfamily of transcription factors [2, 5]. Consistent with the pleiotropic effects of glucocorticoids, the hGR is ubiquitously expressed in all human tissues and cells, and is necessary for life after birth [6]. The hGR protein is encoded by exons 2–9 of the hGR gene (located on chromosome 5) and is composed of four distinct regions: the amino-terminal domain (NTD), the DNA-binding domain (DBD), the hinge region and the ligand-binding domain (LBD) [2, 5]. Alternative splicing of hGR precursor mRNA gives rise to 5 hGR protein subtypes that have been termed hGRα, hGRβ, hGRγ, hGR-A and hGR-P. An additional cohort of eight receptor proteins (hGRα-A, hGRα-B, hGRα-C1, hGRα-C2, hGRα-C3, hGRα-D1, hGRα-D2, and hGRα-D3) is produced by alternative translation initiation from hGR mRNA [2, 5, 7]. hGRα-A is the classic full-length 777-amino acid receptor that is generated from the first translation initiation codon [2, 5, 7]. The other hGRα isoforms have progressively shorter NTDs, possess both common and unique properties and may differentially transduce the glucocorticoid signal to target tissues depending on their selective relative expression and inherent activities. All translational isoforms have similar affinity for the ligand and ability to bind to DNA, consistent with the presence of a common LBD. However, they display differences in their subcellular distribution, with hGRα-D residing constitutively in the nucleus of cells, a fact that indicates that sequences in the NTD of the hGR may play an important role in nuclear translocation, nuclear export and/or cytoplasmic retention of the receptor. In addition, these translational isoforms display significant differences in their ability to regulate gene expression, with the hGRα-C isoforms being the most active, and the hGRα-D subtypes being the most “deficient” in their ability to transactivate glucocorticoid-responsive genes [7]. Recent evidence suggests that hGRα-C3 displays higher transcriptional activity than the other hGRα isoforms, owing to increased recruitment of coactivators at the promoter regions of target genes [8]. In addition to the hGR isoforms generated by alternative splicing or alternative initiation of translation, four novel receptor variants with multiple amino acid replacements/truncation have been described [hGR NS-1, hGR DL-1, hGR-S1 and hGR-S1 (-349A)] [9, 10]. Their functional role remains to be elucidated.

The hGR regulates gene expression by either transcriptional activation (transactivation) or transcriptional repression (transrepression). Prior to binding to glucocorticoids, the hGR resides mostly in the cytoplasm of cells as part of a large multiprotein complex [2, 11]. Upon ligand-induced activation, the receptor undergoes conformational changes that result in dissociation from this multiprotein complex and translocation into the nucleus, where it binds to glucocorticoid-response elements (GREs) in the promoter region of target genes [2, 11, 12]. The latter contain hexamer domains in an inverted palindrome arrangement separated by 3 base pairs in the regulatory regions of target genes and regulate their expression positively or negatively through interaction with coactivators [12] or corepressors [2, 11], respectively. Glucocorticoids may mediate anti-inflammatory effects via direct binding of hGR to evolutionarily conserved negative GREs (nGREs), which contain an inverted tetrameric palindrome separated by 0–2 base pairs that is distinct from the classic GREs [13]. The ligand-activated hGR can also modulate gene expression independently of DNA-binding, by interacting with other transcription factors, such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), p53 and signal transducers and activators of transcription (STATs). The interaction of hGR with the pro-inflammatory transcription factors NF-κB and AP-1 inhibits their activity and accounts for the major anti-inflammatory and immunosuppressive effects of glucocorticoids [3]. Although the transcriptional activity of hGR is primarily governed by ligand binding, post-translational modifications also play important roles. These covalent changes include methylation, acetylation, nitrosylation, sumoylation, ubiquitination and phosphorylation, and may affect receptor stability, subcellular localization, as well as the interaction of hGR with other proteins [11].

In addition to the above-described genomic actions, glucocorticoids can induce some effects via the GR within seconds or minutes. The non-genomic rapid glucocorticoid actions appear to be mediated by membrane bound GRs, which trigger the activation of kinase signaling pathways, thus influencing many CNS and other tissue functions [14]. On the other hand, the MR functions as the 2nd glucocorticoid receptor in some tissues (e.g. limbic structure of the brain and adipose tissue), which do not express the glucocorticoid-inactivating 11β-hydroxysteroid dehydrogenase 2, and cooperates with the classic GR to regulate expression of common and/or distinct target genes [15].

The cellular response to glucocorticoids displays profound variability both in magnitude and in specificity of action [2, 11]. Multiple mechanisms exist to generate diversity, as well as specificity in the response to glucocorticoids, such as pre-receptor ligand metabolism, receptor isoform expression, and receptor-, tissue-, and cell type-specific factors. Furthermore, recent findings from in vitro and in vivo studies have demonstrated the important new role of old molecules, such as the serum- and glucocorticoid-inducible kinase 1 (SGK1) [16, 17] and FK506 -binding protein 51 (FKBP5) [18, 19], in tissue sensitivity to glucocorticoids and associated pathologic conditions. In addition to protein-protein interactions, tissue responsiveness to glucocorticoids has become more complicated since the identification and functional characterization of hGR polymorphisms [20‐24]. Interestingly, MR polymorphisms may also play some roles in tissue glucocorticoid sensitivity [25]. Any of the above-described molecular mechanisms may lead to alterations in tissue sensitivity to glucocorticoids, which may take the form of glucocorticoid resistance or glucocorticoid hypersensitivity and may be associated with significant morbidity (Table 1) [26]. This review summarizes the recent advances in the molecular mechanisms underlying tissue sensitivity to glucocorticoids, with particular emphasis on novel mutations and new information on the circadian rhythm of tissue sensitivity to glucocorticoids, and ligand-induced repression of the glucocorticoid receptor.