eMedicine Specialties > Pediatrics: General Medicine > Endocrinology

Primary Generalized Glucocorticoid Resistance

Author: Evangelia Charmandari, MD, MRCP, MSc, Pediatric and Adolescent Endocrinologist, Senior Investigator, Division of Endocrinology and Metabolism, Biomedical Research Foundation of the Academy of Athens, Greece
Coauthor(s): Tomoshige Kino, MD, PhD, Staff Scientist, Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health; George P Chrousos, MD, FAAP, MACP, MACE, Professor and Chair, Department of Pediatrics, Athens University Medical School
Contributor Information and Disclosures

Updated: Jul 31, 2008

Introduction

Background

Glucocorticoids are steroid hormones synthesized and secreted by the adrenal cortex, largely under the control of the hypothalamic-pituitary-adrenal [HPA] axis.^1,2,3 They regulate various 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.^1,2 Approximately 20% of the genes expressed in human leukocytes are regulated positively or negatively by glucocorticoids.⁴ In addition, glucocorticoids are among the most widely prescribed drugs worldwide and are primarily used as anti-inflammatory and immunosuppressive agents but are also used as chemotherapeutic agents because of their role in induction of apoptosis.

Abnormalities in glucocorticoid sensitivity can be divided into 2 major categories: glucocorticoid resistance and glucocorticoid 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.^5,6 Complete glucocorticoid resistance is not compatible with life, as evidenced by the fact 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.⁷ Resistance syndromes have also been described for the mineralocorticoid,⁸ androgen,⁹ estrogen,¹⁰ progesterone,³ vitamin D, and thyroid hormone receptors.^11,12

Pathophysiology

Molecular mechanisms of glucocorticoid action

At the cellular level, the actions of glucocorticoids are mediated by a 94-kDa intracellular receptor protein, the GR. The GR belongs to the superfamily of steroid/thyroid/retinoic acid receptor proteins that function as ligand-dependent transcription factors (see Media files 1-2).¹³

The molecular structure of GR is similar to that of other steroid receptors and is composed 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 molecules, nuclear translocation, and receptor dimerization (see Media files 1-2).^13,14,15,16

The human glucocorticoid receptor (hGR) complementary DNA (cDNA) was isolated by expression cloning in 1985.¹⁷ 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 α and β. hGRα 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, hGRα mostly resides in the cytoplasm of cells as part of a hetero-oligomeric complex, which contains chaperon heat shock proteins (hsps) 90, 70, and 50, as well as other proteins.^18,19 hsp90 regulates ligand binding, as well as cytoplasmic retention of hGRα by exposing the ligand-binding site and masking the 2 nuclear localization sequences (NLS), NL1 and NL2, which are located adjacent to the DNA-binding domain (DBD) and in the ligand-binding domain (LBD) of the receptor, respectively.

Upon ligand-induced activation, the receptor dissociates from this multiprotein complex and translocates into the nucleus.^18,20 Within the nucleus, the receptor binds as a homodimer to GREs in the promoter regions of target genes and regulates their expression positively or negatively, depending on GRE sequence and promoter context.^19,21 Alternatively, the ligand-activated hGRα can also modulate gene expression independently of DNA-binding, by interacting possibly as a monomer with other transcription factors, such as activator protein-1 (AP-1), nuclear factor-κB (NF-κB), p53, and signal transducers and activators of transcription (STATs).^22,23,24,25 Following transcriptional activation or inhibition of glucocorticoid-responsive genes, hGRα dissociates from the ligand and has a lower affinity for binding to GREs. The unliganded hGRα remains within the nucleus for a considerable length of time and is then exported to the cytoplasm; both within the nucleus and within the cytoplasm, the hGRα may be recycled, degraded, or both in the proteasome (see Media file 3).²⁶

To initiate transcription, hGRα uses its transcriptional activation domains, AF-1 and AF-2, as surfaces to interact with nuclear receptor coactivators (p160, p300/cyclic adenosine monophosphate [CAMP]–response element–binding protein (CBP) and p300/CBP-associated factor [p/CAF]) and chromatin-remodeling complexes (switching sucrose nonfermenting [SWI/SNF] and the vitamin D receptor–interacting protein [DRIP]/thyroid hormone receptor-associated protein [TRAP]).^{27,28,29,30,31} The p160 coactivators, such as the steroid receptor coactivator 1 (SRC1) and the glucocorticoid receptor-interacting protein 1 (GRIP1), interact directly with both the AF-1 of hGRα through their carboxyl-terminal domain and the AF-2 of hGRα through multiple amphipathic LXXLL signature motifs located in their nuclear receptor-binding (NRB) domain.³² They also have histone acetyltransferase activity, which promotes chromatin decondensation and allows the transcription initiation complex of the RNA-polymerase II and its ancillary components to initiate and promote transcription (see Media file 4).^27,28,29,30

Alterations in any of the molecular mechanisms of hGRα action described above may lead to alterations in tissue sensitivity to glucocorticoids, which may take the form of resistance or hypersensitivity and may be associated with significant morbidity.^33,34,35,36 One such condition that the authors have extensively investigated is primary generalized glucocorticoid resistance.³⁶

The hGRβ isoform is also ubiquitously expressed in tissues, albeit at lower concentrations than hGRα.³⁷ In contradistinction to hGRα, hGRβ primarily resides in the nucleus of cells independently of the presence of ligand, does not bind glucocorticoids, and is transcriptionally inactive.^37,38 hGRβ functions as a dominant negative inhibitor of hGRα transcriptional activity and inhibits hGRα–mediated transactivation of many target genes in a dose-dependent manner.³⁹ The ability of hGRβ to antagonize the function of hGRα suggests that hGRβ may play a role in regulating target tissue sensitivity to glucocorticoids.^{40,41,42,43,44}

Increased expression of hGRβ has been documented in generalized and tissue-specific glucocorticoid resistance and leads to a reduction in the ability of hGRα to bind to GREs.^40,41 Therefore, an imbalance in hGRα and hGRβ expression may underlie the pathogenesis of several clinical conditions associated with glucocorticoid resistance, such as rheumatoid arthritis, systemic lupus erythematosus, or ulcerative colitis.⁴⁵

Molecular mechanisms of primary generalized glucocorticoid resistance

The molecular basis of primary generalized glucocorticoid resistance has been ascribed to mutations in the hGR gene, which impair one or more of the molecular mechanisms of hGR action, thereby altering tissue sensitivity to glucocorticoids. Inactivating mutations within the ligand-binding domain or the DNA-binding domain of the receptor and a 4-base pair deletion at the 3'-boundary of exon 6 of the gene have been described in 5 kindreds and 5 sporadic cases.^{46,5,6,47,48,49,50,51,52,53,54,55,56,57,58,59,36,60}  The molecular defects elucidated in the reported cases are summarized in the table below, although the corresponding mutations in the hGR gene are also shown in Media file 5. Most of these mutations (7 of 10) are heterozygous, which suggests that complete loss-of-function of the receptor is incompatible with life.

The authors have identified most hGR mutations associated with primary generalized glucocorticoid resistance and have systematically investigated the molecular mechanisms through which these various natural hGR mutants affect glucocorticoid signal transduction in almost all reported cases. The mechanisms studied included (1) the transcriptional activity of the mutant receptors; (2) the ability of the mutant receptors to exert a dominant negative effect on the wild-type receptor; (3) the affinity of the mutant receptors for the ligand; (4) the subcellular localization of the mutant receptors and their nuclear translocation following exposure to the ligand; (5) the ability of the mutant receptors to bind to GREs; (6) the interaction of the mutant receptors with the GRIP1 coactivator, which belongs to the p160 family of nuclear receptor coactivators and plays an important role in the hGRa-mediated transactivation of glucocorticoid-responsive genes; and (7) the motility of the mutant receptors within the nucleus of living cells.^{6,24,48,49,51,50,33,55,56,57,58,61,59,60,36,62}  

Mutations of the Human Glucocorticoid Receptor Gene Causing Primary Generalized Glucocorticoid Resistance

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In transient transfection assays, all mutant receptors demonstrated variable reduction in their ability to transactivate the glucocorticoid-responsive mouse mammary tumor virus (MMTV) promoter in response to dexamethasone compared with the wild-type receptor; the most severe impairment was observed in cases of R477H (undetectable), I559N (minimal/undetectable), V571A (decreased by 50-fold) and D641V (minimal) mutations.^{49,50,51,52,53,54,55,56,48,58,59,60} Furthermore, the mutant receptors hGRαI559N, hGRαF737L, hGRαI747M, and hGRαL773P exerted a dominant negative effect on the wild-type receptor. The latter might have contributed to manifestation of the disease at the heterozygote state.^{49,53,56,58,60}

Dexamethasone-binding studies showed a variable reduction in the affinity of the mutant receptors for the ligand, with the most severe reduction observed in the cases of I559N (undetectable), I747M (undetectable), and V571A (6-fold) mutations.^{49,51,50,52,53,54,55,56,60,59,57,58} The only mutant receptor that demonstrated normal affinity for the ligand was hGRαR477H, in which the mutation is located at the DBD of the receptor.⁵⁹

The decreased affinity of the mutant receptors for the ligand most likely reflects the location of the mutations in the LBD of hGRα (see Media file 5). The structure of the hGR LBD contains 12 α-helices and 4 small β-strands that fold into a 3-layer helical domain.^63,64 Helices 1 and 3 form one side of a helical sandwich, and helices 7 and 10 form the other side. The middle layer of helices (helices 4, 5, 8, and 9) are present in the top half but not in the bottom half of the protein. This arrangement of helices creates a cavity in the bottom half of the LBD, where the agonist molecule is bound (see Media file 6). Helix 12, which plays an essential function in ligand-dependent activation, adopts the so-called "agonist bound" conformation, where it packs against helices 3, 4 and 10 as an integrated part of the domain structure. Following helix 12, an extended strand forms a conserved β sheet with a β strand between helices 8 and 9. This C-terminal β strand appears to play an important role in receptor activation by stabilizing helix 12 in the active conformation.⁶⁵

Deletion of the last few residues that form the β strand lead to alterations in hormone-binding specificity and significant reduction in the receptor-mediated transactivation of target genes, suggesting that the C-terminal region of hGRα, downstream of helix 12, is essential for ligand-binding specificity and agonist potential, although it does not appear to confer differential hormone-binding capacities to the receptor.^64,65

The authors next studied the subcellular localization and nuclear translocation of the wild-type and mutant receptors in HeLa cells by creating green fluorescent protein-fused constructs of the receptors. In the absence of dexamethasone, hGRα was primarily localized in the cytoplasm of cells. Addition of dexamethasone (10^-6 M) resulted in translocation of the wild-type receptor into the nucleus within 12 minutes. The pathologic mutant receptors were also predominantly observed in the cytoplasm of cells in the absence of ligand, except for the mutant receptors hGRαV729I and hGRαF737L, which were localized both in the cytoplasm and in the nucleus of cells.

Exposure to the same concentration of dexamethasone induced a slow translocation of the mutant receptors into the nucleus, which ranged from 20 minutes (R477H) to 180 minutes (I559N and F737L).^{49,50,51,52,53,54,55,56,48,58,59,60} These findings indicate that all hGR mutations affect the nucleocytoplasmic shuttling of hGRα, probably through impairment of the NL1 and/or NL2 function. Impairment of the NL1 function may arise as a result of the decreased affinity for the ligand, which may prevent a proper ligand-induced allosteric conformation of the receptor and, hence, a normal interaction between NL1 and components of the importin system.⁶⁶ Alternatively, impairment of the NL2 function may specifically depend on the conformation of the LBD induced by the ligand and could also be due to the mutations.⁶⁶ Differential binding of the mutant and wild-type receptors to hsps, which partially inactivate NL1 and NL2, may also contribute to the differences observed between the times required for entry into the nucleus.^14,67,68

Unlike the wild-type and most mutant receptors, the mutant receptors hGRαV729I and hGRαF737L were localized both in the cytoplasm and in the nucleus of cells in the absence of ligand. The β isoform of hGR, which has a defective, non–ligand-binding LBD, as well as all hGRα mutants that lack their LBD, also primarily localize in the nucleus of cells.³³ This suggests that the LBD of hGRα plays an important role in the cytoplasmic retention of the receptor in the absence of ligand. Alternatively, defective mechanisms that may relate to delayed nuclear export, such as the calreticulin export pathway and certain motifs in the DBD that function as nuclear export signals, might account for the nuclear localization of the unliganded hGRαV729I and hGRαF737L,^69,70 an effect that might be similar to the nuclear retention mechanism of hGRβ.³³  

The authors investigated the ability of the mutant receptors to bind to DNA in electrophoretic mobility shift assays and chromatin immunoprecipitation assays.^{49,50,51,52,53,54,55,56,48,58,59,60} The wild-type and all mutant receptors in which the mutations were located in the LBD of hGRα preserved their ability to bind to DNA. The only mutant receptor that failed to bind to DNA was the hGRαR477H, in which the mutation is located at the C-terminal zinc finger of the DBD of the receptor.⁵⁹ A major function of the C-terminal zinc finger of the DBD of hGRα is to contribute to receptor homodimerization, a prerequisite for potent receptor binding to GREs and efficient transactivation of glucocorticoid-responsive genes.^71,72 This function is achieved by a group of 5 amino acids in the N-terminal knuckle of the C-terminal zinc finger of the receptor, known as the D loop or dimerization domain. Point mutations in the DBD of the GR may abolish DNA-binding, resulting in silencing of transcriptional activation, although they may not affect the ability of the mutant receptors to transrepress AP-1-, NF-κB-, and/or other targeted gene-dependent transcription, possibly through protein-to-protein interactions and/or tethering of other cofactors to the transcriptional machinery.^72,73,74,75

To determine whether the mutant receptors displayed an abnormal interaction with the p160 coactivators, the authors investigated the interaction between the mutant receptors and the GRIP1 coactivator in a glutathione-S-transferase pull-down assay. GRIP1 contains 2 sites that bind to steroid receptors. One site, the NRB, is located at the amino terminus of the protein and interacts with the AF-2 of hGRα in a ligand-dependent fashion. The other site is located at the carboxyl-terminus of the protein and binds to the AF-1 of hGRα in a ligand-independent fashion.^76,77,78 The wild-type and most mutant receptors bound to full-length GRIP1 and the carboxyl-terminal fragment of GRIP1 but not to the NRB fragment of GRIP1, suggesting that these mutant receptors interact with the GRIP1 coactivator in vitro only through their AF-1. Exceptions represented the mutant receptors hGRαR477H, which interacted with GRIP1 both through its AF-1 and AF-2, and hGRαI559N, which did not interact with either fragment of GRIP1.^{49,50,51,52,53,54,55,56,48,58,59,60}

Helix 12 plays a critical role in the formation of both the ligand-binding pocket and the AF-2 surface that facilitates interaction with coactivators. Upon ligand-binding, the receptor undergoes major conformational changes that alter the position of helix 11 and helix 12 and generate an interaction surface that allows coactivators to bind to AF-2 through their LXXLL motifs.⁶⁴ In the agonist-bound conformational state of hGR LBD, helix 12 adopts a position over the ligand-binding pocket, which allows coactivators to interact within the coactivator cavity, thus forming a transcriptionally active receptor.

Alternatively, binding to an antagonist induces structural changes that lead to loss of the helical structure in the C-terminal portion of helix 11 and movement of helix 12 over the ligand-binding pocket, a position that prevents coactivator binding and enables corepressor binding (see Media file 6).⁶⁴ The presence of various mutations in the LBD of hGRα likely influences the orientation of helix 12, either by preventing contact between this helix and the ligand or by displacing it from its active position.^79,80,81 These findings indicate that the hGR mutant receptors may form a defective complex with GRIP1, which is partially or completely ineffective. Furthermore, the mutant receptors may also display an abnormal interaction with other AF-2-associated proteins, such as the p300/CBP cointegrators and components of the DRIP/TRAP complex.^27,28,29,30

Using fluorescence recovery after photobleaching (FRAP) analysis, all hGR pathologic mutant receptors had defective transcriptional activity and dynamic motility defects inside the nucleus of living cells.⁶² In the presence of dexamethasone, these mutants displayed a curtailed 50% recovery time (t^1/2) after photobleaching and, hence, significantly increased intranuclear motility and decreased chromatin retention. The t^1/2 values of the mutants correlated positively with their transcriptional activities and depended on the hGR domain affected. Thus, mutant hGRs possess dynamic motility defects in the nucleus, possibly caused by their inability to properly interact with all key partner nuclear molecules necessary for full activation of glucocorticoid-responsive genes. The motility defect of the mutant receptors is directly proportional to their transactivation defect, indicating that the former is a good overall index of functionality.⁶²

Finally, the authors examined the association between the location of the known mutations in the crystal structure of the LBD of hGR and the molecular mechanisms through which these mutations impaired glucocorticoid signal transduction. Media file 7 illustrates the location of the known hGR mutations in the agonist and antagonist form of the LBD of hGRα. Two mutations (ie, I559N and V571A) are located within helix 5, whereas 4 mutations (ie, V729I, F737L, I747M, and L773P) are located close to helix 11 and helix 12. All mutations within the LBD of the receptor were shown to affect the affinity of the receptor for the ligand; however, this effect was more pronounced in the cases of I559N and V571A mutations located in helix 5 of the receptor. Nuclear translocation was more delayed in the cases of I559N, V729I, and F737L mutations, implicating mostly helix 5, helix 10, and helix 11. All mutations within the LBD of the receptor affected the in vitro interaction of the receptor with the GRIP1 coactivator but preserved their ability to bind to DNA. The one mutation (R477H) identified in the DBD of the receptor primarily impaired the ability of the receptor to bind to GREs. The fact that most mutant receptors interacted with the GRIP1 coactivator in vitro only through their AF-1 domain highlights the importance of helix 10, helix 11, and helix 12 of the LBD of the receptor in facilitating the formation of the AF-2 surface that interacts with coactivators.

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 primarily occurs in children and women.

Clinical

History

Clinical manifestations of primary generalized glucocorticoid resistance

Primary generalized glucocorticoid resistance is a rare familial or sporadic genetic condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids.^{46,5,6,47,48,59} The latter leads to activation of the HPA axis and compensatory elevations in circulating cortisol and adrenocorticotropic hormone (ACTH) concentrations, which maintain circadian rhythmicity and appropriate responsiveness to stressors. The excess ACTH secretion results in adrenal hyperplasia, and increased production of adrenal steroids with mineralocorticoid activity (eg, cortisol, deoxycorticosterone [DOC], corticosterone) and/or androgenic activity(eg, androstenedione, dehydroepiandrosterone [DHEA], DHEA-sulfate [DHEAS]).^{46,5,6,47,48,59} See Media file 8.

The clinical presentation of primary generalized glucocorticoid resistance is summarized below and relates to the pathophysiologic alterations depicted in Media file 8. Generally, clinical manifestations of glucocorticoid deficiency have not been reported in subjects affected with the condition, with the exception of chronic fatigue, which might indicate inadequate compensation by the increased cortisol concentrations in certain resistant target tissues, such as the CNS or the skeletal muscles. Symptoms and signs of mineralocorticoid excess, such as hypertension and hypokalemic alkalosis, have been reported in many affected subjects and are attributed to the elevated concentrations of cortisol, DOC, and corticosterone.^{46,5,6,47,48,49,50,51,52,53,54,55,56,48,58,59,60}

The increased concentrations of adrenal androgens in subjects with the condition result in manifestations of androgen excess, such as ambiguous genitalia, in a child with 46,XX chromosomes at birth and gonadotropin-independent precocious puberty in children of either gender. Acne, hirsutism, and infertility occurs in both sexes; male-pattern hair loss, menstrual irregularities, and oligo-anovulation occur in females; and oligospermia occurs in males.⁶⁰ The impaired fertility in both sexes is most likely due to the feedback inhibition of gonadotropin secretion by the elevated androgen concentrations. Finally, the increased corticotropin-releasing hormone (CRH) and ACTH concentrations are likely to account for the profound anxiety described in some cases and may predispose affected subjects to the development of intratesticular adrenal rests and/or ACTH-secreting pituitary adenomas.⁴⁹

• Clinical presentation
• Apparently normal glucocorticoid function
• Asymptomatic
• Chronic fatigue (glucocorticoid deficiency)
• Mineralocorticoid excess
• Hypertension
• Hypokalemic alkalosis
• Androgen excess
• Children - Ambiguous genitalia at birth (only case of ambiguous genitalia in a child with 46,XX karyotype who also harbored a heterozygous mutation of the 21-hydroxylase gene), premature adrenarche, precocious puberty
• Females - Acne, hirsutism, male-pattern hair loss, menstrual irregularities, oligo-anovulation, infertility
• Males - Acne, hirsutism, oligospermia, adrenal rests in the testes, infertility
• Increased HPA axis activity (CRH/ACTH hypersecretion) - Anxiety
• Adrenal rests
• Diagnostic Evaluation
• Absence of clinical features of Cushing syndrome
• Normal or elevated plasma ACTH concentrations
• Elevated plasma cortisol concentrations
• Increased 24-hour urinary free cortisol excretion
• Normal circadian and stress-induced pattern of cortisol and ACTH secretion
• Resistance of the HPA axis to dexamethasone suppression
• Thymidine incorporation assays - Increased resistance to dexamethasone-induced suppression of phytohemagglutinin-stimulated thymidine incorporation compared with control subjects
• Dexamethasone-binding assays - Decreased affinity of the glucocorticoid receptor for the ligand compared with control subjects
• Molecular studies - Mutations/deletions of the glucocorticoid receptor

The clinical spectrum is broad, ranging from severe to mild forms; many patients are asymptomatic, displaying only biochemical alterations.^{46,5,6,47,48,49,50,51,52,53,54,55,56,48,58,59,60}  This variable clinical phenotype is likely to be due to (1) variations in the glucocorticoid, mineralocorticoid, or androgen receptor–signaling pathways; (2) variations in the degree of tissue sensitivity to glucocorticoids, mineralocorticoids, and/or androgens; (3) variations in the activity of key hormone-inactivating or hormone-activating enzymes, such as the 11β-hydroxysteroid dehydrogenase⁸² and 5α-reductase;⁸³ and (4) other genetic or epigenetic factors, such as insulin resistance and visceral obesity.⁶

Causes

See Pathophysiology.

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