Evaluation of Glucocorticoid Sensitivity and Its Potential Clinical Applicability

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Horm Res 2009;71:305309

DOI: 10.1159/000223413

Evaluation of Glucocorticoid Sensitivityand Its Potential Clinical Applicability

Carlos Alberto Longuia, b Cludia Dutra Costantin Fariab

aMolecular Medicine Laboratory, Physiology Department of Faculty of Medical Sciences of Santa Casa de

So Paulo, and bPediatric Endocrinology Unit, Pediatric Department of Irmandade da Santa Casa de Misericrdia

de So Paulo, So Paulo, Brazil

particularly related to abnormal tissue-speciic GC resis-tance or GC hypersensitivity [1].

GC are largely employed as a therapy, as a substituteor cortisol, or because o their anti-inlammatory e-ects, when given in supra-physiologic doses [2]. In cer-tain cell types, cell death by apoptosis can also be reachedwith therapeutic doses o GC, which is a relevant eect inthe treatment o neoplastic disorders.

Clinical response to GC is extremely variable. This isrelated to the GC type, dose and schedule, and to the dis-

ease or cell type to be targeted; however, it is also depen-dent on actors modulating each individuals GC sensi-tivity [3].

Interestingly, patients with GC-dependent disordersare those usually presenting tissue-speciic resistance,such as in arthritis and asthma [45]. Concomitance oinadequate inlammatory control and excessive eectson the pituitary, bone and other tissues are common ind-ings in these individuals, suggesting variability in corti-sol metabolism and action in dierent tissues [67]. Ev-ery individuals cellular GC sensitivity is an inluentialactor, along with the GC receptor (GR) numbers, regula-

tion o splice or translational GR variants, mutations and/or polymorphisms in the GR gene, and the availability ocoactors as important variables [8].

This reinorces the need or recognition o individualvariation, and organ- or tissue-speciic aspects modulat-ing GC sensitivity. The recognition o this proile canhave relevant implications or GC therapy.

Key Words

Glucocorticoid sensitivity Glucocorticoid receptor

Dexamethasone suppression test

Abstract

Glucocorticoids (GC) play an important role in physiologic

and pathophysiologic adaptive responses to stress. The ma-

jority of these effects are mediated by the GC receptors (GR).

GC sensitivity largely depends of the amount of available GR,

and their ability to bind the GC-responsive element and/orother nuclear transcription factors, leading to modulation of

the expression of GC target genes. Clinical conditions of tis-

sue-specific GC resistance or GC hypersensitivity have been

described in several diseases, such as chronic inflammatory

and autoimmune conditions, and in visceral obesity, such as

metabolic syndrome. Several in vivo and in vitro methods

have been described, allowing the evaluation and quantita-

tion of GC sensitivity. The recognition of these parameters

has improved our comprehension of the mechanisms in-

volved in those diseases, with potential implications for the

diagnosis and therapy of such abnormalities.

Copyright 2009 S. Karger AG, Basel

Introduction

Glucocorticoids (GC) are important modulators in avariety o physiologic systems, and are also a key compo-nent involved in the pathophysiology o several diseases

Received: November 17, 2008

Accepted: April 24, 2009

Published online: June 6, 2009

HORMONERESEARCH

Carlos Alberto LonguiRua Proessor Artur Ramos 96, 20. andarJardim Paulistano

01454-010 So Paulo (Brazil)Tel./Fax +55 11 3222 0628, E-Mail c [email protected]

2009 S. Karger AG, Basel03010163/09/07160305$26.00/0

Accessible online at:www.karger.com/hre


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Longui /FariaHorm Res 2009;71:305309306

Considering that the GC eects are predominantly

mediated by the GR, one o the major determinants o GCresponsivity is the intracellular density o the active iso-orm GR, which is in agreement with the correlationobserved between GC sensitivity and the amount o GRdetermined by several in vitro assays [9].

In this review, we briely described the multiplicity oactors involved in the signaling cascade o GC actionand their inluence on GC sensitivity. We also report onthe experience gained by our group in the developmento in vivo and in vitro methods, which can be helpul inquantiying GC sensitivity in both physiologic condi-tions, such as physical exercise, and pathologic condi-

tions, such as obesity and rheumatoid arthritis.

Modulators of GC Sensitivity

Signiicant dierences have been observed in GC sen-sitivity among species, individuals, tissues, or even di-erent cells at a dierent cell-cycle phase [1]. Several mod-ulators o GC sensitivity have already identiied, such as(1) bioactive GC availability, in luenced by cortisol-bind-ing globulin and by the activity o 11-hydroxysteroiddehydrogenase types 1 type 2; (2) the number, hormonal

ainity and phosphorylation pattern o GR; (3) nucleartranslocation and the ability to bind and activate the GC-responsive element; (4) interrelationship with the tran-scription actors, leading to modulation o the transcrip-tion o GC target genes [1013] (ig. 1).

In pathologic conditions, such as trauma, acute orchronic diseases, hypoglycemia and behavioral disor-ders, cortisol production increases several times [1416].

In inlammatory diseases, cytokines such as interleu-

kins 1 and 6, tumor necrosis actor- and leukemia in-hibitory actor are substantially increased, rendering aGC insensitivity and an interplay among modulatorsrom the endocrine and immune systems [17].

GR mutations and polymorphisms are also determi-nants of GC sensitivity, inf luencing the binding affinity tothe ligand and the expression rate of the GR gene [18]. Re-sistance to GC can be generalized or tissue specific. Gen-eralized resistance includes the hypothalamic and the pi-tuitary regions (with a consequent failure in the negativefeedback), elevated ACTH concentrations, and increasedcortisol secretion. Excessive ACTH stimulation deter-

mines higher androgen and mineralocorticoid productionwith a variable clinical presentation of virilization and saltretention [18]. Clinical manifestations of GC resistanceand a description of the mutations in the gene encodingGR are presented in tables 1 and 2, respectively.

Generalized GC hypersensitivity is a rare phenome-non and it is poorly characterized. Iida et al. [19] irst re-ported a patient with symptoms o Cushings syndrome,despite hypocortisolemia. The molecular etiology o GChypersensitivity has not been ully clarifed, but 2 singlenucleotide polymorphisms o the GR gene seem to playan important role in determining hypersensitivity [20].

The N363S polymorphism has been associated with in-creased sensitivity to GC, lower bone mineral density,and increased BMI [21]; however, other reports ound noassociation with BMI [22]. Another polymorphism (BclIpolymorphism), recently identifed as a C]G nucleotidechange, associated the G allele with increased sensitivityto GC [23]. In men, the BclI haplotype was associatedwith a 34% higher risk o cardiovascular disease [24].

GRNuclear translocation

Ability to bind and activate GRE

Ability to bind nuclear transcription

factors

Pre-receptor modulators:

HPA axis

Bioactive GC availability:

CBG activity

11 -HSD type 1 and 2 activities

activity

Pre-receptor modulators:

GR mutations and polymorphisms:

Number of GR

Hormonal affinity

Phosphorylation pattern

GR modulators:

Fig. 1. Modulators o GS. HPA = Hypotha-lamic-pituitary-adrenal axis; CBG = corti-sol-binding globulin; 11-HSD = 11 hy-droxy-steroid dehydrogenase; GRE = GC-responsive elements (target genes).


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In vivo Evaluation of GC Sensitivity

The in vivo models used to study GC sensitivity wereinitially directed to detect GC eects, such as salt reten-tion, inluence on the carbohydrate metabolism, andanti-inlammatory and pro-apoptotic actions [2527].NF-B and activator protein 1 also interere with these

GC eects, and should be measured simultaneously[28].

The standard in vivo evaluation is represented by thedetermination of cortisol suppression after dexametha-sone. High doses of dexamethasone have been used for thediagnosis of Cushing syndrome. On the other hand, dosesvarying from 20 to 80 g/kg/day invariably suppress cor-tisol in all normal subjects, preventing its use in the recog-nition of normal individual GC sensitivity [29]. We previ-ously reported the use of dexamethasone in a single doseof 75 g/m2 p.o. in normal and in obese children [30], al-lowing the detection of GC sensitivity in a physiologic

range. In order to exclude the interference on dexametha-sone metabolism during the first passage through the liv-er [31], we subsequently described the use of i.v. bolus ad-ministration of dexamethasone in a dose of 20g/m2 [32].This very low dose of i.v. dexamethasone is a useful meth-od for in vivo establishment of GC sensitivity.

We also tested the applicability o this i.v. very-low-dose dexamethasone suppression test (VLD-DST) in

some stressul conditions. In normal individuals submit-ted to intensive and prolonged physical training, low bas-al cortisol levels associated with lower cortisol reduc-tion ater the i.v. VLD-DST are observed ater the train-ing program. Together, these observations suggest loweractivity o the HPA axis with a reduction in the pituitarynegative eedback to dexamethasone, possibly relecting

a more systemic reduction in GC sensitivity [33].

In vitro Evaluation of GC Sensitivity

The methods to perorm in vitro measurements o GCsensitivity include determination o glucose or aminoacid concentrations and transport, protein synthesis, and

Table 1. Clinical maniestations o GC resistance

Clinical presentation

Apparently normal GC unctionAsymptomaticChronic atigue

Mineralocorticoid excessHypertensionHypokalemic alkalosis

Androgen excessChildren: ambiguous genitalia at birth, premature

adrenarche, precocious pubertyFemales: acne, hirsutism, male-pattern hair loss,

menstrual irregularities, oligo-anovulation, inertilityMales: acne, hirsutism, oligospermia, adrenal rests in the

testes, inertilityIncreased HPA axis activity (CRH/ACTH hypersecretion)

AnxietyAdrenal rests

CRH = Corticotropin-releasing hormone; ACTH = adreno-corticotropic hormone. Modiied rom reerence 18.

Table 2. Molecular basis and phenotype o GC resistance

Mutation position

cDNA Amino acid Phenotype

1922 (A]T) 641 (D]V) hypertensionhypokalemic alkalosis

4-bp deletion inexon-intron 6

hirsutismmale-pattern hair lossmenstrual irregularities

2185 (G]A) 729 (V]I) precocious puberty hyperandrogenism

1676 (T]A) 559 (I]N) hypertensionoligospermiainertility

1430 (G]A) 477 (R ]H) hirsutismatiguehypertension

2035 (G]A) 679 (G]S) hirsutismatiguehypertension

1712 (T]C) 571 (V]A) ambiguous genitaliahypertensionhypokalemiahyperandrogenism

2241 (T]G) 747 (I]M) cystic acnehirsutismoligo-amenorrhea

2318 (T]C) 773 (L]P) atigueanxietyacnehirsutismhypertension

2209 (T]C) 737 (F]L) hypertensionhypokalemia

Modiied rom reerence 18.


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Longui /FariaHorm Res 2009;71:305309308

RNA expression assays. Measurements o cell survival,cell prolierations, or cell death are also useul [34].

Considering that GC action is predominantly medi-ated by GR [35], several qualitative and semi-quantita-tive assays were previously described. Initial techniquesemployed GR-binding capability in intact cells, allowing

quantitation o bioactive cytosolic/nuclear GR [36]. Tis-sue-speciic GR gene expression was also described bymeasuring GR-cDNA obtained rom several tissues sam-ples or by Northern blotting analyses [37]. Homologousdownregulation o GR is possible by Western blotting as-says employing speciic anti-GR antibodies [38].

We recently described a method o absolute quantita-tion o GR gene expression by real-time PCR [39]. Thismethod is accurate and reproducible, with intra- and in-ter-assay coeicients o variation o 2 and 7%, respec-tively. It employs a standardized cell line in the construc-tion o standard curves, and a normalizing gene (BCR).

We also showed a relationship between GR expressionand the GC sensitivity measured by the intravenousVLD-DST, especially those cases in which high GR ex-pression was observed (unpublished data).

The eects o intensive and prolonged exercise on geneexpression were also evaluated by real-time PCR, show-ing a signiicant reduction in GR and a global decrease

in the expression o in lammatory genes o the IKK/IB/NF-B pathway, accompanied by a reduction in severalinterleukins [40].

GR gene expression in patients with rheumatoid ar-thritis showed an inverse correlation with GC sensitivity,suggesting that an upregulation o GR is present in these

patients, possibly related to a post-receptor phenomenon[41].

Additional clinical applicability should be evaluated,employing a combination o methods described in thisshort review. This can contribute to the diagnosis andtherapeutic schedule o several diseases presenting vari-able states o GC sensitivity, like tissue-speciic GC resis-tance described in asthma, rheumatoid arthritis, andlymphoblastic cells [45, 38]. In clinical practice, GCis widely used to treat diseases (e.g. asthma, chronicinammation, prevention o rejection o organ trans-plants) as well as replacement therapy. It is known that

GC eects vary considerably between patients. Some pa-tients respond to therapeutical administration o GC, butalso develop side eects; while others need a very highdose to establish clinical eects. This dose should be ad-justed to a patient need, considering the GC sensitivity,in such a way that it is therapeutically eective but doesnot cause side eects.

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