Excerpt from Glucocorticoid Resistance Syndrome

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Background

Glucocorticoids are steroid hormones synthesized and secreted by the adrenal cortex largely under the control of the hypothalamus and pituitary (the hypothalamic-pituitary-adrenal [HPA] axis) (Munck, 1984; Clark, 1992; Simpson, 1995). They regulate a variety of biologic processes and exert profound influences on many physiologic functions by virtue of their diverse roles in growth, development, and maintenance of basal and stress-related homeostasis (Munck, 1984; Clark, 1992). In addition, glucocorticoids are among the most widely prescribed drugs worldwide, used primarily as anti-inflammatory and immunosuppressive agents, but also as chemotherapeutic agents because of their role in induction of apoptosis.

Abnormalities in glucocorticoid sensitivity can be divided into 2 major groups: resistance and hypersensitivity. Target tissue resistance to glucocorticoids is characterized by the inability of target tissues to respond to these hormones and can be generalized or tissue-specific, transient or permanent, partial or complete, and compensated or noncompensated (Chrousos, 1982; Chrousos, 1993). Complete glucocorticoid resistance is not compatible with life, given that absence of functional glucocorticoid receptors (GRs) in GR -/- knockout mice leads to severe neonatal respiratory distress syndrome and death within a few hours after birth (Cole, 1995). Resistance syndromes have also been described for the mineralocorticoid (Zennaro, 1996), androgen (McPhaul, 1993), estrogen (Smith, 1994), progesterone (Simpson, 1998), vitamin D, and thyroid hormone receptors (Hewison, 1993; Refetoff, 1993).

Pathophysiology

Molecular mechanisms of glucocorticoid action

At the cellular level, the actions of glucocorticoids are mediated by a 94 kDa intracellular receptor protein, the glucocorticoid receptor (GR). The GR belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins that function as ligand-dependent transcription factors (Carson-Jurica, 1990) (see Images 1-2).

The molecular structure of GR is similar to that of other steroid receptors and is comprised of 3 functional domains: (1) a poorly conserved amino terminal domain (NTD), which contains a major transactivation domain termed activation function (AF)–1; (2) a central, highly conserved DNA-binding domain, which contains 2 zinc finger motifs through which it binds to specific DNA sequences in the promoter region of target genes, the glucocorticoid response elements (GREs); and (3) a carboxyl terminal ligand-binding domain, which contains a second transactivation domain AF-2, as well as sequences important for interaction with heat shock protein (hsp) molecules, nuclear translocation, and receptor dimerization (Carson-Jurica, 1990; Picard, 1987; Hollenberg, 1988; Dalman, 1991) (see Images 1-2).

The human glucocorticoid receptor (hGR) cDNA was isolated by expression cloning in 1985 (Hollenberg, 1985). The hGR gene consists of 9 exons and is located on chromosome 5. Alternative splicing in exon 9 generates 2 highly homologous receptor isoforms, termed alpha and beta. hGR alpha is ubiquitously expressed in almost all human tissues and cells and represents the classic hGR that functions as a ligand-dependent transcription factor. In the absence of ligand, hGR alpha resides mostly in the cytoplasm of cells as part of a large multiprotein complex, which consists of the receptor polypeptide, 2 molecules of hsp90, and several other proteins (Bamberger, 1996). The hsp90 molecules are thought to sequester hGR alpha in the cytoplasm of cells by maintaining the receptor in a conformation that masks or inactivates its nuclear localization signals (NLSs) but empowers it to interact with glucocorticoids.

Upon hormone binding, the receptor undergoes an allosteric change, which results in dissociation from hsp90 and other proteins and unmasking or activation of the NLSs. In its new conformation, the activated ligand-bound GR translocates into the nucleus, where it binds as a homodimer to GREs located in the promoter region of target genes (Bamberger, 1996). The hGR alpha then communicates with the basal transcription machinery and regulates the expression of glucocorticoid-responsive genes positively or negatively, depending on the GRE sequence and promoter context (Bamberger, 1996). The receptor can also modulate gene expression independently of GRE binding by physically interacting with other transcription factors, such as activator protein (AP)–1 and nuclear factor (NF)–kB (Jonat, 1990; Scheinman, 1995).

The ability of ligand-bound hGR alpha to transactivate steroid-responsive genes depends on the presence of coactivators, nucleoproteins with chromatin remodeling and other enzymatic activities, which are attracted to the promoter region of target genes via the AF-1 and AF-2 of hGR alpha (McKenna, 1999; McKenna, 2002) (see Image 3). Several families of coactivators have been described, including the p160 coactivators, such as the steroid receptor coactivator 1 (SRC1) and the GR-interacting protein 1 (GRIP1), the p300/CREB-binding protein (CBP) cointegrators, the p300/CBP-associated protein (PCAF), the switching/sucrose nonfermenting (SWI/SNF) complex, and the vitamin D receptor–interacting protein/thyroid hormone–associated protein (DRIP/TRAP) complex (Refetoff, 1993; Carson-Jurica, 1990; Picard, 1987; Hollenberg, 1988).

The GRIP1 coactivator contains 2 sites that bind to steroid receptors. One site, the nuclear receptor binding (NRB) site, is located at the amino terminus, between amino acids 542 and 745, and contains 3 LXXLL signature motifs, through which it interacts with the AF-2 of hGR alpha in a ligand-dependent fashion. The other site is located at the carboxyl terminus, between amino acids 1121 and 1250, and binds to the AF-1 of hGR alpha in a ligand-independent fashion.

The p160, CBP/p300, and PCAF proteins have histone acetyltransferase activity, which promotes chromatin decondensation, allowing the transcriptional preinitiation complex of RNA-polymerase II and its ancillary components to access the GRE-promoter region and initiate transcription (Auboeuf, 2002; Hittelman, 1999).

The hGR beta isoform is also ubiquitously expressed in tissues, albeit at lower concentrations than hGR alpha (de Castro, 1996). In contradistinction to hGR alpha, hGR beta resides primarily in the nucleus of cells independently of the presence of ligand, does not bind glucocorticoids or antiglucocorticoids, does not activate glucocorticoid-responsive genes, and is transcriptionally inactive (de Castro, 1996; Oakley, 1997). hGR beta functions as a dominant negative inhibitor of hGR alpha activity and inhibits hGR alpha–mediated transactivation of many target genes in a dose-dependent manner (Oakley, 1999). The ability of hGR beta to antagonize the function of hGR alpha suggests that hGR beta may play a role in regulating target tissue sensitivity to glucocorticoids (Leung, 1997; Shahidi, 1999; Longui, 2000; Hauk, 2002; Hauk, 2000).

Increased expression of hGR beta has been documented in generalized and tissue-specific glucocorticoid resistance and leads to a reduction in the ability of hGR alpha to bind to GREs (Leung, 1997; Shahidi, 1999). Therefore, an imbalance in hGR alpha and hGR beta expression may underlie the pathogenesis of several clinical conditions associated with glucocorticoid resistance, such as rheumatoid arthritis, systemic lupus erythematosus, or ulcerative colitis (Chrousos, 1995).

Molecular mechanisms of glucocorticoid resistance

The molecular basis of glucocorticoid resistance in several families and sporadic cases has been ascribed to mutations in the hGR alpha gene, which impair one or more of the molecular mechanisms of GR function, thus altering tissue sensitivity to glucocorticoids. Since glucocorticoid resistance was first described and investigated in detail (Chrousos, 1982; Vingerhoeds, 1976), more than 10 kindreds and sporadic cases with the condition have been reported.

Abnormalities of several hGR alpha characteristics, including cell concentrations, affinity for glucocorticoids, and translocation into the nucleus, have been associated with this condition (Hurley, 1991; Karl, 1993; Malchoff, 1993; Karl, 1996; Kino, Stauber, 2001; Vottero, 2002; Ruiz, 2001; Mendonca, 2002). The molecular defects that have been elucidated in the reported cases are summarized in the Table, while the corresponding mutations in the hGR alpha gene are shown in both the Table and in Image 4.

The propositus of the original kindred was homozygous for a single point mutation at nucleotide position 2054, which results in a nonconservative amino acid substitution at position 641, replacing aspartic acid with valine (Hurley, 1991). Compared to the wild-type receptor, this mutant receptor results in decreased transactivation of the glucocorticoid-responsive mouse mammary tumor virus (MMTV) promoter and a 3-fold reduction in the affinity for dexamethasone (Hurley, 1991). In the absence of ligand, the mutant receptor is primarily localized in the cytoplasm of cells. Exposure to dexamethasone (10⁶ M) induces a slow translocation into the nucleus, which takes 22 minutes as opposed to 12 minutes required for nuclear translocation of the wild-type receptor. Finally, the mutant receptor interacts with the amino terminal but not with the carboxyl terminal fragment or full-length GRIP1 in vitro (Charmandari, 2002).

The propositus of the second family was heterozygous for a 4-base deletion at the 3'-boundary of exon and intron 6. The deletion removed a donor splice site in 1 allele, affecting the last 2 bases of the exon 6 and the first 2 bases of the intron 6. This resulted in complete ablation of the expression of one of the hGR alpha alleles and a decrease in GR alpha protein by 50% in affected members of the family (Karl, 1993).

The propositus of the third kindred was homozygous for a point mutation at nucleotide position 2317, which results in substitution of valine for isoleucine at amino acid 729 of the ligand-binding domain of hGR alpha (Malchoff, 1993). This mutation results in decreased transcriptional activity of the receptor and a 4-fold reduction in the affinity for dexamethasone (Malchoff, 1993). The mutant receptor is localized primarily in the nucleus of cells in the absence of ligand, further translocation from the cytoplasm into the nucleus requires longer (120 min) exposure to dexamethasone (10⁶ M), and the receptor demonstrates a weak ligand-dependent interaction with the full-length and carboxyl terminal fragment but not with the amino terminal fragment of GRIP1 in vitro (Charmandari, 2002).

The first sporadic case of glucocorticoid resistance was due to a de novo, germ-line, heterozygous mutation at nucleotide position 1808, resulting in substitution of isoleucine for asparagine at amino acid 559 in the hormone-binding domain of hGR alpha. This mutation reduces the transcriptional activity of hGR alpha significantly and has been associated with the development of an adrenocorticotropic hormone (ACTH)–secreting adenoma (Karl, 1996).

Although the affinity for ligand was preserved in the patient studied, a 50% decrease was observed in the hGR binding sites (Karl, 1996). Furthermore, the mutant receptor has a markedly delayed nuclear translocation (180 min) and a dominant negative activity upon the wild-type receptor, ie, it decreases the transcriptional activity of hGR alpha in a dose-dependent manner (Kino, Stauber, 2001). The latter may account for manifestation of the disease at the heterozygotic state. No interaction exists between the mutant receptor and GRIP1 (Charmandari, 2002). This mutant receptor has been associated with the development of Cushing disease due to an ACTH-secreting adenoma (Karl, 1996).

The fifth and sixth cases of glucocorticoid resistance were due to heterozygous mutations at nucleotide positions 1430 and 2035, resulting respectively in substitution of arginine for histidine at amino acid 477 and glycine for serine at amino acid 679 (Ruiz, 2001). The former mutation is located in the second zinc finger of the DNA-binding domain. This mutant receptor has no transactivation activity because of impaired binding to GREs but has the same affinity for ligand as the wild-type receptor. The latter mutation is located in the ligand-binding domain, outside the ligand-binding pocket, and results in a 50% reduction both in the transcriptional activity and the ligand-binding affinity of the receptor (Ruiz, 2001).

The propositus of the seventh case was homozygous for a point mutation at nucleotide position 1844, which results in a valine-to-alanine substitution at amino acid 571 in the ligand-binding domain of hGR alpha (Mendonca, 2002). This mutation causes up to 50-fold decrease in the transcriptional activity of the receptor and a 6-fold reduction in the affinity for ligand (Mendonca, 2002). The nuclear translocation of the mutant receptor is delayed (25 min), while its interaction with the GRIP1 coactivator occurs mostly via its AF-1 domain (Charmandari, 2002).

The eighth case of glucocorticoid resistance was due to a heterozygous mutation at nucleotide position 2373, which causes an isoleucine-to-methionine substitution at amino acid 747 in the ligand-binding domain of the receptor (Vottero, 2002). This mutation is located at the carboxyl terminus of the ligand-binding domain, close to helix 12, which plays a pivotal role in the formation of AF-2, a domain that interacts with p160 and other coactivators. The mutant receptor has a 20- to 30-fold decrease in the transactivation of the MMTV promoter, 2-fold reduction in the affinity for dexamethasone and delayed nuclear translocation. It also exerts a dominant negative effect upon the wild-type hGR alpha and interacts with the GRIP1 coactivator in vitro only through its intact AF-1 domain. Overexpression of GRIP1 restores the transcriptional activity and reverses the dominant negative activity of the mutant upon the wild-type receptor (Vottero, 2002).

Finally, the ninth case of glucocorticoid resistance was due to a heterozygous mutation at nucleotide position 2318, which results in substitution of leucine by praline at amino acid 773 in the ligand-binding domain of the receptor (Charmandari, 2005). Compared with the wild-type hGR alpha, the mutant receptor demonstrates a 50% reduction in its ability to transactivate the MMTV promoter. Furthermore, it exerts a dominant negative effect upon the wild-type receptor, has a 2.6-fold reduction in the affinity for ligand, shows delayed nuclear translocation, and although it preserves its ability to bind to DNA, it displays abnormal interaction with the GRIP1 coactivators in vitro.

The authors have also demonstrated that (1) the ligand-binding domains of the mutant receptors hGRaI559N, hGRaV571A, hGRaD641V, hGRaV729I, and hGRaI747M have decreased intrinsic transcriptional activity; (2) hGRaV571A, hGRaD641V, and hGRaV729I do not exert a dominant negative effect on the transcriptional activity of hGR alpha; and (3) all 5 mutant receptors hGRaI559N, hGRaV571A, hGRaD641V, hGRaV729I, and hGRaI747M preserve their ability to bind to DNA (Charmandari, 2002). Therefore, the process through which the above hGR alpha mutant receptors impair the physiologic mechanisms of glucocorticoid action at the molecular level is multifactorial, and involves impaired ability to bind ligand, aberrant nucleocytoplasmic trafficking, and abnormal interaction with the p160 coactivators. These variable functional defects of the mutant receptors upon the glucocorticoid-signaling pathway may explain the genetic transmission and the variable clinical phenotype of glucocorticoid resistance.

In addition to mutations in the hGR alpha gene, steroid receptor coactivator defects may also account for generalized glucocorticoid resistance and/or resistance to other steroid hormones. In 1999, New et al described 2 sisters with multiple partial steroid resistance whose clinical and biochemical findings were largely consistent with isolated glucocorticoid resistance (New, 1999; Chrousos, 1999). However, the paradoxic absence of hyperandrogenic manifestations necessitated further evaluation, which also revealed resistance to mineralocorticoids, estrogen, and androgens but not to vitamin D or thyroid hormones (New, 1999; Chrousos, 1999).

Mutations of the Human Glucocorticoid Receptor Gene Causing Glucocorticoid Resistance

*LBD indicated ligand-binding domain.

Frequency

United States

Glucocorticoid resistance is rare.

International

This condition is rare internationally.

Mortality/Morbidity

Cardiovascular morbidity and mortality is increased if not treated.

Sex

Hyperandrogenism is a problem in children and women primarily.

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