Low- versus high-baseline epinephrine output shapes opposite innate cytokine profiles: presence of Lewis- and Fischer-like neurohormonal immune phenotypes in humans?

Immunogenetic mechanisms operating within the immune system are known to influence cytokine profiles and disease susceptibility. Yet the role of the individual's neurohormonal background in these processes remains undefined. Hormonal imbalances are documented in immune-related diseases, but it is unclear whether this represents a secondary phenomenon or a primary "defect" related to specific neurohormonal immune phenotype(s). We report that in a large subpopulation of healthy humans the baseline epinephrine output (but not cortisol and sex steroid hormones) correlated inversely with proinflammatory and positively with anti-inflammatory cytokine production. Thus, low vs high epinephrine excretors had a 2- to 5-fold higher TNF-alpha and IL-12 production but 2-fold lower IL-10 production induced by LPS ex vivo. In alternative settings, we found low baseline levels and profoundly blunted stress-induced epinephrine responses but high TNF-alpha levels in Lewis vs Fischer inbred rats. Additionally, isoproterenol, a beta adrenoreceptor agonist suppressed LPS-induced TNF-alpha production, with more pronounced effect in Lewis than in Fischer rats. In human monocytes, epinephrine and the beta(2) adrenoreceptor agonist fenoterol potently inhibited LPS-induced TNF-alpha and IL-12, but stimulated IL-10 production. The order of potency for hormones able to inhibit IL-12 production ex vivo was: epinephrine > norepinephrine > or = 1,25-(OH)(2) vitamin D(3) > hydrocortisone. This indicates that baseline epinephrine conditions cytokine responsiveness and through this mechanism intrinsic hypo- or hyperactive adrenal medullas in some individuals may shape opposite cytokine profiles. Since Lewis and Fischer rats have opposite susceptibility to experimental immunological diseases, this suggests that the parallel human phenotypes could be linked to differing responsiveness and susceptibility to infections and immune/inflammatory-related conditions.

A novel point mutation in the amino terminal domain of the human glucocorticoid receptor (hGR) gene enhancing hGR-mediated gene expression.

CONTEXT: Interindividual variations in glucocorticoid sensitivity have been associated with manifestations of cortisol excess or deficiency and may be partly explained by polymorphisms in the human glucocorticoid receptor (hGR) gene. We studied a 43-yr-old female, who presented with manifestations consistent with tissue-selective glucocorticoid hypersensitivity. We detected a novel, single, heterozygous nucleotide (G --> C) substitution at position 1201 (exon 2) of the hGR gene, which resulted in aspartic acid to histidine substitution at amino acid position 401 in the amino-terminal domain of the hGRalpha. We investigated the molecular mechanisms of action of the natural mutant receptor hGRalphaD401H. METHODS-RESULTS: Compared with the wild-type hGRalpha, the mutant receptor hGRalphaD401H demonstrated a 2.4-fold increase in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone but had similar affinity for the ligand (dissociation constant = 6.2 +/- 0.6 vs. 6.1 +/- 0.6 nm) and time to nuclear translocation (14.75 +/- 0.25 vs. 14.25 +/- 1.13 min). The mutant receptor hGRalphaD401H did not exert a dominant positive or negative effect upon the wild-type receptor, it preserved its ability to bind to glucocorticoid response elements, and displayed a normal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. CONCLUSIONS: The mutant receptor hGRalphaD401H enhances the transcriptional activity of glucocorticoid-responsive genes. The presence of the D401H mutation may predispose subjects to obesity, hypertension, and other manifestations of the metabolic syndrome.

A novel point mutation in helix 11 of the ligand-binding domain of the human glucocorticoid receptor gene causing generalized glucocorticoid resistance.

BACKGROUND: Generalized glucocorticoid resistance is a rare condition characterized by partial, end-organ insensitivity to glucocorticoids, compensatory elevations in adrenocorticotropic hormone and cortisol secretion, and increased production of adrenal steroids with androgenic and/or mineralocorticoid activity. We have identified a new case of glucocorticoid resistance caused by a novel mutation of the human glucocorticoid receptor (hGR) gene and studied the molecular mechanisms through which the mutant receptor impairs glucocorticoid signal transduction. METHODS AND RESULTS: We identified a novel, single, heterozygous nucleotide (T --> C) substitution at position 2209 (exon 9alpha) of the hGR gene, which resulted in phenylalanine (F) to leucine (L) substitution at amino acid position 737 within helix 11 of the ligand-binding domain of the protein. Compared with the wild-type receptor, the mutant receptor hGRalphaF737L demonstrated a significant ligand-exposure time-dependent decrease in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone and displayed a 2-fold reduction in the affinity for ligand, a 12-fold delay in nuclear translocation, and an abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. The mutant receptor preserved its ability to bind to DNA and exerted a dominant-negative effect on the wild-type hGRalpha only after a short duration of exposure to the ligand. CONCLUSIONS: The mutant receptor hGRalphaF737L causes generalized glucocorticoid resistance because of decreased affinity for the ligand, marked delay in nuclear translocation, and/or abnormal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. These findings confirm the importance of the C terminus of the ligand-binding domain of the receptor in conferring transactivational activity.

Functional characterization of the natural human glucocorticoid receptor (hGR) mutants hGRalphaR477H and hGRalphaG679S associated with generalized glucocorticoid resistance.

BACKGROUND: Glucocorticoid resistance is often a result of mutations in the human glucocorticoid receptor alpha (hGRalpha) gene, which impair one or more of hGRalpha's functions. We investigated the molecular mechanisms through which two previously described mutant receptors, hGRalphaR477H and hGRalphaG679S, with amino acid substitutions in the DNA- and ligand-binding domains, respectively, affect glucocorticoid signal transduction. METHODS AND RESULTS: In transient transfection assays, hGRalphaR477H displayed no transcriptional activity, whereas hGRalphaG679S showed a 55% reduction in its ability to stimulate the transcription of the glucocorticoid-responsive mouse mammary tumor virus promoter in response to dexamethasone compared with the wild-type hGRalpha. Neither hGRalphaR477H nor hGRalphaG679S exerted a dominant negative effect upon the wild-type receptor. Dexamethasone binding assays showed that hGRalphaR477H preserved normal affinity for the ligand, whereas hGRalphaG679S displayed a 2-fold reduction compared with hGRalpha. Nuclear translocation studies confirmed predominantly cytoplasmic localization of the mutant receptors in the absence of ligand. Exposure to dexamethasone resulted in slower translocation of hGRalphaR477H (25 min) and hGRalphaG679S (30 min) into the nucleus than the wild-type hGRalpha (12 min). In chromatin immunoprecipitation assays in cells stably transfected with the mouse mammary tumor virus promoter, hGRalphaR477H did not bind to glucocorticoid-response elements, whereas hGRalphaG679S preserved its ability to bind to glucocorticoid-response elements. Finally, in glutathione-S-transferase pull-down assays, hGRalphaG679S interacted with the glucocorticoid receptor-interacting protein 1 coactivator in vitro only through its activation function (AF)-1, unlike the hGRalphaR477H and hGRalpha, which interacted with the glucocorticoid receptor-interacting protein 1 through both their AF-1 and AF-2. CONCLUSIONS: The natural mutants hGRalphaR477H and hGRalphaG679S cause generalized glucocorticoid resistance by affecting different functions of the glucocorticoid receptor, which span the cascade of the hGR signaling system.

A novel point mutation in the ligand-binding domain (LBD) of the human glucocorticoid receptor (hGR) causing generalized glucocorticoid resistance: the importance of the C terminus of hGR LBD in conferring transactivational activity.

Glucocorticoid resistance is a rare, familial or sporadic condition characterized by partial end-organ insensitivity to glucocorticoids. The clinical spectrum of the condition is broad, ranging from completely asymptomatic to severe hyperandrogenism and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance has been ascribed to mutations in the human glucocorticoid receptor-alpha (hGRalpha) gene, which impair one or more of the molecular mechanisms of GR action, thus altering tissue sensitivity to glucocorticoids. We identified a new case of generalized glucocorticoid resistance in a young woman who presented with a long-standing history of fatigue, anxiety, hyperandrogenism, and hypertension. The disease was caused by a novel, heterozygous mutation (T-->C) at nucleotide position 2318 (exon 9) of the hGRalpha gene, which resulted in substitution of leucine by proline at amino acid position 773 in the ligand-binding domain of the receptor. We systematically investigated the molecular mechanisms through which the natural hGRalphaL773P mutant impaired glucocorticoid signal transduction. Compared with the wild-type hGRalpha, hGRalphaL773P demonstrated a 2-fold reduction in the ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter, exerted a dominant negative effect on the wild-type receptor, had a 2.6-fold reduction in the affinity for ligand, showed delayed nuclear translocation (30 vs. 12 min), and, although it preserved its ability to bind to DNA, displayed an abnormal interaction with the GR-interacting protein 1 coactivator in vitro. We conclude that the carboxyl terminus of the ligand-binding domain of hGRalpha is extremely important in conferring transactivational activity by altering multiple functions of this composite transcription factor.

Five mutations of mitochondrial DNA polymerase-gamma (POLG) are not a prevalent etiology for spontaneous 46,XX primary ovarian insufficiency.

The study objective was to determine if mutations in mitochondrial DNA polymerase gamma (POLG) are associated with spontaneous 46,XX primary ovarian insufficiency (sPOI) using restriction fragment length polymorphism analysis of genomic DNA. Of 201 women with 46,XX sPOI analyzed, we found only one case (0.5%, 95% confidence interval 0-3%) of heterozygosity for a POLG mutation, suggesting that this is not a common genetic etiology for this form of infertility.

Investigation of the human stem cell factor KIT ligand gene, KITLG, in women with 46,XX spontaneous premature ovarian failure.

OBJECTIVE: To investigate mutations in the human KIT ligand gene (KITLG) gene as a mechanism of 46,XX spontaneous premature ovarian failure. The human KIT ligand gene, known also as human stem cell factor, is the ligand of the c-kit transmembrane tyrosine kinase receptor (KIT). This ligand-receptor interaction is known to play important roles in mouse germ cell migration and proliferation. DESIGN: Cross-sectional study. SETTING: Clinical research center. PATIENT(S): Forty women with 46,XX spontaneous premature ovarian failure. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Single-stranded conformational polymorphism analysis and DNA sequencing. RESULT(S): We found one nucleotide change of the KITLG coding region (811G-->T) that led to an alteration of the amino acid composition of the KITLG protein in one Caucasian patient (Asp210Tyr). However, we found the same alteration in two normal control Caucasian samples. Three nucleotide substitutions were found in the noncoding exon of KITLG (exon 10). We also identified two intronic polymorphisms. Thus, we did not identify a single significant mutation in the coding region of the KITLG gene in any of 40 patients (upper 95% confidence limit is 7.2%). CONCLUSION(S): Mutations in the coding regions of the KITLG gene appear not to be a common cause of 46,XX spontaneous premature ovarian failure in North American women.