Glucocorticoids (GCs) are essential for life exerting their effects in virtually every tissue and organ in the body (1). These hormones are major effectors of basal and stress related homeostasis, influence cardiovascular function and carbohydrate, protein and fat metabolism, and regulate the immune/inflammatoryreaction. They also activate the central nervous system and in addition, they participate in the development and basal functions of several organs and systems. Glucocorticoids exert these effects by binding to specific intracellular receptor proteins, the glucocorticoid receptors (GRs). By binding to the GRs, GCs influence most of the metabolic effects via interaction of the receptor complex with glucocorticoid- responsive elements (GREs) in the promoters of target genes; on the other hand, their anti-inflammatory/immunosoppressive actions are mostly exerted via protein-protein interaction with positive regulators of growth and inflammation, such as AP-1 and NF-kB, respectively. Through these mechanisms, the active fractions, represented by the albumin-bound and free cortisol, exert a negative feedback on both hypothalamic corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) secretion and inhibit pituitary ACTH secretion itself, therefore, controlling the activity of the hypothalamic-pituitary-adrenal (HPA) axis.
Abnormalities of steroid sensitivity can be divided into two major groups: resistance and hypersensitivity. Steroid resistance syndromes have been described for GCs (2-5), mineralocorticoids (6, 7), androgens (8), estrogens (9), and progesterone (10, 11). Resistance to both vitamin D and thyroid hormones receptors has also been described (12-14). Target tissue resistance to steroid hormones implies inability of these tissues to respond to these hormones (15). This resistance can be transient or permanent, partial or complete and compensated or noncompensated. Complete glucocorticoid resistance is incompatible with life, as demonstrated in a study where the absence of functional GR in GR -/- knock-out mice led to death within a few hours after birth because of severe neonatal respiratory distress syndrome. The lungs of the newborns were atelectatic and their livers had a reduced capacity to activate gluconeogenic enzymes (16). These animals had a normal number of adrenal chromaffin cells in their adrenal medullae, but they lack the adrenaline-synthesizing enzyme PNMT and secretogranin II (17).
When partial steroid resistance occurs, the HPA axis is reset at higher levels of ACTH and cortisol. The latter compensate for the insensitivity of tissues to glucocorticoids; however, the increased ACTH levels result in increased secretion of glucocorticoid precursors with mineralocorticoid activity (deoxycorticosterone, DOC, and corticosterone) and adrenal androgens. The clinical spectrum of the primary cortisol resistance syndrome is broad varying from completely asymptomatic to fatigue, hyperandrogenism and hypertension with or without hypokalemic alkalosis (3, 15).
Over 10 kindreds and a few sporadic cases suffering from congenital generalized glucocorticoid resistance have been described to date and the molecular mechanisms of their resistance have been analyzed in some of them (18-23).
To act on their target tissues, glucocorticoids first interact with specific intracellular receptor proteins which are members of the nuclear receptor superfamily (Fig. 1), including mineralocorticoids (with which they share the highest homology), androgens, progestins, estrogens, vitamin D, thyroid hormone, retinoic acids, and a growing number of “orphan” receptors, for most of which the specific ligand has not been identified as yet (24, 25). The sequence of GR cDNA was first published in 1985 and, since then, two alternative splicing products of the same gene located on chromosome 5 were described (26) (Fig. 2): the classic, active receptor called GRα, and the dominant negative isoform GRβ. They share the first 727 amino acids and differ just in the last 50 and 15 amino acids, respectively. Therefore, the first 8 exons of the GR gene containing the 5' noncoding and coding sequences are common to both receptor isoform cDNAs, while exons 9α and 9β, containing the coding and 3' noncoding sequences, are specific for GRα and GRβ, respectively.
Figure 2. Structure of the hGR gene and its products. Alternative splicing events, #1 (default splicing pathway) and #2 (alternative splicing pathway) generate two different hGR messages, which differ in size due to the use of alternative polyadenylation signals. Translation of the messages produces two isoforms of the glucocorticoid receptor, hGRα and hGRβ respectively, which have identical structure through amino acid 727 but then diverge. hGRα is 777 amino acids long, has a molecular weight of about 98 kDa, while hGRβ, is 742 amino acids long, and has a molecular weight of about 94 kDa. Functional domains and the putative sites are indicated below the linearized GR protein. Boxes and lines represent exons and introns, respectively.
The modular structure of GR is similar to those of other members of the nuclear receptor superfamily, and consists of three functional domains: a) the amino terminal, "immunogenic" domain, containing a strong independent transactivation domain (AF-1 or t1), which is important for regulation of target gene expression (27); b) the middle, DNA-binding domain (DBD) which consists of two "zinc fingers" necessary for receptor dimerization, nuclear translocation (NLS1), binding to GREs in the promoters of responsive genes, and GRE-mediated transactivation (28); c) and the carboxyl-terminal ligand-binding domain (LBL) (24, 29), which, in addition to ligand binding, contains sequences important for heat shock protein 90 (hsp 90) binding, nuclear translocation (NLS2), receptor dimerization, and a second transactivation domain (AF-2 or t2), as well as corepressor domains which are important for silencing of the receptor in the absence of the hormone (30).
Recently, several transcription factor "coregulators", which alter the effect of nuclear hormone receptors on the transcription of target genes, have been described (31); these include the CBP/P300 and SRC-1 (p160) families of "coactivators" (32, 33) and the NcoR/RIP13 and SMRT/TRAC families of "corepressors" (34, 35).
In the absence of hormone, GRα resides predominately in the cytoplasm of cells in a multiprotein complex consisting of the receptor polypeptide, two molecules of hsp90, one molecule of hsp70, and one molecule of hsp 56, which is an immunophilin of the cyclosporin-, FK506- and rapamycin-binding classes (36). GRα binds with glucocorticoids and transactivates or transrepresses glucocorticoid-responsive promoters. After binding, the receptor-ligand complex undergoes a conformational change, thus releasing the hsp complex and homodimerizing with another activated GRα The activated GRα monomer or dimer interact with the importin system and translocate via the nuclear pore into the nucleus, where they regulate gene expression. They do so by binding with GREs in the presence of coactivators or via protein-protein interaction interacting with other transcription factors and by preventing them from - or less frequently by potentiating them- in exerting their effects on their own target genes (Fig. 3A). Most GREs are associated with enhanced transcription; however there are several examples of "negative" GREs (nGREs), as described in the pro-opiomelanocortin (37), osteocalcin (38) and prolactin promoters (39), which are associated with repression of transcription. The GRα as a monomer, on the other hand, modulates the transcription rates of non-GRE-containing genes by interacting with nuclear transcription factors including AP-1 (40), NF-kB (41) and STAT5 (42) (Fig. 3B).
Figure 3a. Putative mechanisms of action of hGRα and hGRβ.In the unliganded state, the classic glucocorticoid receptor α resides predominantly in the cytoplasm as part of a heteromeric complex including at least 5 molecules of heat shock proteins. After binding to the hormone, the GRα molecule changes its conformation and is released by the heat shock proteins. Then as a monomer or as a dimer with another hormone-activated receptor molecule, translocates into the cell nucleus, where it regulates the transcriptional activities of target genes. hGRβ is unable to bind glucocorticoids and is transcriptionally inactive, but may have a cell-specific dominant negative effect on GRα primarily on GRE-mediated actions. A GRE consensus sequence (15bp) is shown.
Figure 3b. Nuclear actions of GRα and GRβ. After entering the nucleus, ligand-bound GRα influences transcription of target genes by different mechanisms: it may activate transcription by binding to GRE as a homodimer; as a heterodimer with GRβ it may have diminished ability to transactivate a GRE-containing gene and, hence, may act as a dominant negative inhibitor. Binding of a GRα homodimer to a negative GRE (nGRE) may also lead to repression; a GRα-GRβ heterodimer may lose the ability to repress a nGRE. Monomeric GRα interacts with other transcription factors such as AP-1 and NF-kB and prevents them from exerting their activities on the promoters of their target genes containing AP-1 and NF-kB sites, respectively. In a similar protein-protein interaction between GRα and Stat-5, GRα acts synergistically with Stat-5 in the activation of Stat5-responsive genes. There is no evidence that GRβ interacts or influences the activity of AP-1 or NF-kB. This could be exerted by binding to GREs or through protein-protein-interaction with other transcription factors.
In contrust to GRα, GRβ does not bind to glucocorticoids and functions as weak dominant inhibitor of GRα. This action is mediated by GRE binding, since no protein-protein interaction has been described. The intracellular localization of GRβ is uncertain (43); our group reported that most GRα and GRβ immunoreactivity of HeLa cells was in the cytosolic fraction when they were in a resting state, while incubation with dexamethasone resulted in the translocation of both isoforms in the nuclear fraction probably under the form of GRα-GRβ heterodimers (44). In contrast, Oakley et al. (45) demonstrated that GRβ resided primarily in the nucleus of transfected cells, independently of hormone treatment, which could be explained by the reduced affinity of GRβ to hsp90 (45). Recently, we found green-fluorescent protein-GRβ fusion hybrids in the nucleus of transfected cells confirming the potential presence of GRβ in the nucleus under certain conditions even in the absence of ligand. The export of GRβ from the nucleus was much slower than that of GRα which could explain its nuclear accumulation (46).