High Affinity Glucocorticoid Receptors Research Articles

The animal and cell models that uncovered FKBP51 as a regulator of glucocorticoid receptor function.

Many New World primates are glucocorticoid-resistant secondary to expression of low affinity glucocorticoid receptors. We identified the role of FKBP51 in hormone responsiveness by showing that multiple cell lines derived from New World primates share the same activities: (1) soluble cell extracts conferred low binding affinity to high affinity glucocorticoid receptors; (2) FK506 increased receptor binding in soluble cell extracts; and (3) cellular FKBP51 was elevated and FKBP52 was lower. Details of these cell lines and their availability are described. Subsequently, we showed that New World primate and human FKBP51 decreased glucocorticoid activity in heterologous COS-7 cell cultures. Future studies using the FKBP51 antagonist SAFit2 in New World primates are proposed.

The Synthesis and Structural Requirements for Measuring Glucocorticoid Receptor Expression In Vivo with (±)-11C-YJH08 PET.

Noninvasive methods to study glucocorticoid receptor (GR) signaling are urgently needed to elaborate the complexity of GR signaling in normal physiology and human disorders, as well as to identify selective GR modulators to treat diseases. Here, we report evidence supporting translational studies with (±)-11C-5-(4-fluorobenzyl)-10-methoxy-2,2,4-trimethyl-2,5-dihydro-1H-chromeno[3,4-f]-quinoline ((±)-11C-YJH08), a radioligand for PET that engages the ligand binding domain on GR. Methods: (±)-11C-YJH08 was synthesized by reacting the phenol precursor with 11C-methyl iodide. The biodistribution was studied in vivo. Specific binding was tested in vivo with adrenalectomy and ligand competition. A library of analogs was synthesized and studied in vitro and in vivo to understand the (±)-11C-YJH08 structure-activity relationship. Rodent dosimetry studies were performed to estimate the human-equivalent doses of (±)-11C-YJH08. Results: (±)-11C-YJH08 was synthesized by reaction of the phenolic precursor with 11C-methyl iodide, giving a radiochemical yield of 51.7% ± 4.7% (decay-corrected to starting 11C-methyl iodide). Specific binding was observed in many tissues, including the brain. An analysis of the (±)-YJH08 structure-activity relationship showed that (R)- and (S)-enantiomers are equally avid for GR by occupying discrete binding modes. A focused chemical screen revealed that the aryl fluoride motif on YJH08 is essential for high-affinity GR binding in vitro, high tissue uptake in vivo, and efficient passage across the blood-brain barrier. Lastly, we performed dosimetry studies on rodents, from which we estimated the human-equivalent doses of (±)-11C-YJH08 to be commensurate with the widely used 11C and 18F tracers. Conclusion: These studies reveal the molecular determinants of a high-affinity and high-selectivity ligand-receptor interaction and support the use of (±)-11C-YJH08 PET to make the first measurements of GR expression in human subjects.

Contribution of Glucocorticoids and Glucocorticoid Receptors to the Regulation of Neurodegenerative Processes

Isolation of glucocorticoids (GCs) from adrenal glands followed by synthesis led rapidly to their first clinical application, about 70 years ago, for treatment of rheumatoid arthritis. To this day GCs are used in diseases that have an inflammatory component. However, their use is carefully monitored because of harmful side effects. GCs are also synonymous with stress and adaptation. In CNS, GC binds and activates high affinity mineralocorticoid receptor (MR) and low affinity glucocorticoid receptor (GR). GR, whose expression is ubiquitous, is only activated when GC levels rise as during circadian peak and in response to stress. Numerous recent studies have yielded important and new insights on the mechanisms concerning pulsatile secretory pattern of GCs as well as various processes that tightly control their synthesis via hypothalamic-pituitary-adrenal (HPA) axis involving regulated release of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) from hypothalamus and pituitary, respectively. GR modulates neuronal functions and viability through both genomic and non-genomic actions, and importantly its transcriptional regulatory activity is tightly locked with GC secretory pattern. There is increasing evidence pointing to involvement of GC-GR in neurodegenerative disorders. Patients with Alzheimer's or Parkinson's or Huntington's disease show chronically high cortisol levels suggesting changes occurring in controls of HPA axis. In experimental models of these diseases, chronic stress or GC treatment was found to exacerbate both the clinical symptoms and neurodegenerative processes. However, recent evidence also shows that GC-GR can exert neuroprotective effects. Thus, for any potential therapeutic strategies in these neurodegenerative diseases we need to understand the precise modifications both in HPA axis and in GR activity and find ways to harness their protective actions.

Optimization of stress response through the nuclear receptor-mediated cortisol signalling network

It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body’s response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress.

Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator

Glucocorticoid receptor (GR) antagonism may be of considerable therapeutic value in stress-related psychopathology such as depression. However, blockade of all GR-dependent processes in the brain will lead to unnecessary and even counteractive effects, such as elevated endogenous cortisol levels. Selective GR modulators are ligands that can act both as agonist and as antagonist and may be used to separate beneficial from harmful treatment effects. We have discovered that the high-affinity GR ligand C108297 is a selective modulator in the rat brain. We first demonstrate that C108297 induces a unique interaction profile between GR and its downstream effector molecules, the nuclear receptor coregulators, compared with the full agonist dexamethasone and the antagonist RU486 (mifepristone). C108297 displays partial agonistic activity for the suppression of hypothalamic corticotropin-releasing hormone (CRH) gene expression and potently enhances GR-dependent memory consolidation of training on an inhibitory avoidance task. In contrast, it lacks agonistic effects on the expression of CRH in the central amygdala and antagonizes GR-mediated reduction in hippocampal neurogenesis after chronic corticosterone exposure. Importantly, the compound does not lead to disinhibition of the hypothalamus-pituitary-adrenal axis. Thus, C108297 represents a class of ligands that has the potential to more selectively abrogate pathogenic GR-dependent processes in the brain, while retaining beneficial aspects of GR signaling.

Photoperiod and stress regulation of corticosteroid receptor, brain-derived neurotrophic factor, and glucose transporter GLUT3 mRNA in the hippocampus of male Siberian hamsters (Phodopus sungorus)

In response to changing day lengths, small photoperiodic rodents have evolved a suite of adaptations to survive the energetic bottlenecks of winter. Among these adaptations are changes in metabolism, adiposity, and energy balance. Whereas hypothalamic and neuroendocrine regulation of these adaptations has been extensively studied, the impact of day length, and interaction of day length and stress, on the energy balance of neurons within the central nervous system remains unspecified. Thus, we exposed male Siberian hamsters (Phodopus sungorus) to either short or long day lengths for 14weeks to induce the full suite of adaptive responses, exposed them to 4h of restraint, and then measured relative mRNA expression in the hippocampus for low- and high-affinity glucocorticoid receptors (glucocorticoid receptor (GR), mineralocorticoid receptor (MR)), brain-derived neurotrophic factor (BDNF), and the neuron-specific glucose transporter GLUT3. Independent of photoperiod, restraint elevated plasma cortisol (CORT) concentrations and reduced expression of GR, MR, and BDNF. Neither restraint nor photoperiod significantly altered GLUT3 expression. Among all groups, plasma cortisol concentrations were negatively correlated with GR and MR expression. MR, BDNF, and GLUT3 levels were positively correlated with one another, even when controlling for photoperiod and CORT. Taken together, these results suggest that, as peripheral energy balance changes across day length in this photoperiodic species, the neurons of the hippocampus do not alter relative gene expression levels of three proteins involved in monitoring neuronal glucose regulation and morphology.

Long-term Survival and Occasional Regression of Distant Melanoma Metastases after Adrenal Metastasectomy

Human melanoma cells express high-affinity glucocorticoid receptors, and adrenalectomy has been shown to have antimelanoma effects in animal models. Long-term regression of distant metastatic melanoma was observed in one patient after bilateral adrenalectomy, prompting a review of adrenalectomy for melanoma metastases performed at this center. A retrospective study in which all patients treated at the Sydney Melanoma Unit and recorded as having adrenal gland metastases between January 1987 and January 2004 were identified and their survival analyzed. One hundred eighty-six patients with adrenal gland metastases were identified. Adrenalectomy was performed in 23 patients; the other 163 patients were treated nonsurgically. The adrenal glands were the sole site of disease in five patients. All symptomatic patients were free of pain after recovery from the surgical procedure. Thirteen patients were rendered clinically and radiologically disease-free by their surgery. There was no postoperative mortality within 30 days. Median overall survival after adrenalectomy was 16 months (2-year survival, 39%), compared with 5 months for patients with documented adrenal metastases treated nonsurgically (P < .00001). In one patient, nonresected visceral metastases elsewhere regressed completely after bilateral adrenalectomy; he remained well and free of disease 80 months after adrenalectomy. Regression of distant visceral metastatic disease also occurred in a second patient after unilateral adrenalectomy. Adrenalectomy for melanoma metastatic to the adrenal gland provides good palliation of symptoms and is associated with prolonged survival in a selected cohort of patients. We report for the first time sustained complete regression of distant metastatic melanoma after bilateral adrenalectomy, suggesting a possible role for adrenal hormones in modifying melanoma progression in certain patients.

Plasticity and Anxiety

This article has no abstract.

Mineralocorticoid Receptors: Distribution and Activation

Mineralocorticoid receptors (MR) bind both mineralocorticoids and glucocorticoids with high affinity (deoxycorticosterone = corticosterone >/= aldosterone = cortisol), and are found in both Na(+) transporting epithelia (e.g. kidney, colon) and nonepithelial tissues (e.g. heart, brain). MR evolved before aldosterone synthase, consistent with their acting in nonepithelial tissues as high affinity glucocorticoid receptors, essentially always occupied by normal levels of endogenous glucocorticoids. In epithelial tissues the enzyme 11beta hydroxysteroid dehydrogenase Type 2 (11betaHSD2) allows aldosterone to selectively activate MR, by converting cortisol to cortisone and NAD to NADH. 11betaHSD2 debulks intracellular cortisol by 90%, to levels approximately 10-fold those of aldosterone, so that when the enzyme is operating most epithelial MR are occupied but not activated by cortisol. When intracellular redox state is changed-by inhibition of 11beta HSD2, generation of reactive oxygen species, or intracellular introduction of oxidised glutathione (GSSG)-cortisol changes from an MR antagonist to an MR agonist. This bivalent activity of cortisol appears to underlie the therapeutic efficacy of MR blockade in heart failure (RALES, EPHESUS) and in essential hypertension, providing a rationale for MR blockade in cardiovascular disease not characterized by elevated aldosterone levels. Its wider (patho)physiologic implications, particularly for neurobiology, remain to be explored.

Long-term survival and disease regression after adrenalectomy for adrenal metastases from melanoma

7540 Background: Human melanoma cells express high affinity glucocorticoid receptors, and adrenalectomy has been shown to have anti-melanoma effects in animal models. We observed long-term regression of metastatic melanoma in two patients following bilateral adrenalectomy, prompting a review of adrenalectomy at the Sydney Melanoma Unit (SMU). Methods: All SMU patients having adrenalectomy for melanoma metastatic to the adrenal gland(s) between January 1987 and June 2001 were identified and examined. Results: 17 patients underwent adrenalectomy for adrenal metastases, 5 females and 12 males, median age 62 years. The adrenal gland(s) were the sole site of disease in 13 patients. Indications were pain (n=4), or the detection of bulky metastases on radiological imaging (n=13). Five patients had bilateral adrenal metastases resected. Significant post-operative morbidity occurred in 2 patients (12%), but there was no post-operative mortality. The median survival of patients after adrenalectomy was 19 months (2 year survival 46%), compared with 4 months for patients with documented adrenal metastases concurrently treated with best supportive care (n=127) (p=0.0001). In two patients non-resected metastases elsewhere regressed following bilateral adrenalectomy, and both remain disease free over three years later. Conclusion: As previously reported, adrenalectomy for adrenal melanoma metastases is associated with prolonged survival in selected patients. We report for the first time sustained complete regression of distant metastatic melanoma following adrenalectomy, suggesting a possible role for adrenal hormones in modifying melanoma in certain patients. No significant financial relationships to disclose.

Long-term survival and disease regression after adrenalectomy for adrenal metastases from melanoma

7540 Background: Human melanoma cells express high affinity glucocorticoid receptors, and adrenalectomy has been shown to have anti-melanoma effects in animal models. We observed long-term regression of metastatic melanoma in two patients following bilateral adrenalectomy, prompting a review of adrenalectomy at the Sydney Melanoma Unit (SMU). Methods: All SMU patients having adrenalectomy for melanoma metastatic to the adrenal gland(s) between January 1987 and June 2001 were identified and examined. Results: 17 patients underwent adrenalectomy for adrenal metastases, 5 females and 12 males, median age 62 years. The adrenal gland(s) were the sole site of disease in 13 patients. Indications were pain (n=4), or the detection of bulky metastases on radiological imaging (n=13). Five patients had bilateral adrenal metastases resected. Significant post-operative morbidity occurred in 2 patients (12%), but there was no post-operative mortality. The median survival of patients after adrenalectomy was 19 months (2 year survival 46%), compared with 4 months for patients with documented adrenal metastases concurrently treated with best supportive care (n=127) (p=0.0001). In two patients non-resected metastases elsewhere regressed following bilateral adrenalectomy, and both remain disease free over three years later. Conclusion: As previously reported, adrenalectomy for adrenal melanoma metastases is associated with prolonged survival in selected patients. We report for the first time sustained complete regression of distant metastatic melanoma following adrenalectomy, suggesting a possible role for adrenal hormones in modifying melanoma in certain patients. No significant financial relationships to disclose.

New steroidal antiinflammatory antedrugs: Methyl 3,20-dioxo-9α-fluoro-11β,17α,21-trihydroxy-1,4-pregnadiene-16α-carboxylate and its 21-O-acyl derivatives

In a continuing effort to increase local to systemic activity ratios of potent steroidal antiinflammatory antedrugs, a series of 21-O-acyl derivatives of methyl 3,20-dioxo-9 alpha-fluoro-11 beta,17 alpha,21-trihydroxy-1,4-pregnadiene-16 alpha-carboxylate, FP16CM, were synthesized. These derivatives were evaluated for antiinflammatory activity and their adverse effects in an acute and semi-chronic croton oil-induced ear edema bioassay. Following a single topical application in the croton oil-induced ear edema bioassay, treatment with all the compounds resulted in dose-dependent inhibition of edema. From these dose-response profiles, the following ID(50) values (nmol/ear resulting in a 50% reduction of edema) were calculated: prednisolone (Pred); 454, FP16CM; 255, 21-acetate (FP16CM-acetyl); 402, 21-propionate (FP16CM-propionyl); 474, 21-valerate (FP16CM-valeryl); 446 and 21-pivalate (FP16CM-pivalyl); 219 nmol. In a 5-day semi-chronic study at the equipotent doses, the novel steroidal antedrugs did not significantly alter body weight gain, thymus weights or plasma corticosterone levels unlike the parent compound Pred. The compounds were assessed for high-affinity glucocorticoid receptor binding and glucocorticoid-mediated inhibition of nitric oxide (NO) generation in an in vitro RAW 264.7 macrophage cell culture system. Binding affinities for cytosolic glucocorticoid receptors were Pred; 85, FP16CM-acetyl; 86, FP16CM-propionyl; 169, FP16CM-valeryl; 149, FP16CM-pivalyl; 126 nM, respectively. Concomitant potencies for inhibition of NO generation by macrophages stimulated with lipopolysaccharide were Pred; 159, FP16CM-acetyl; 377, FP16CM-propionyl; 405, FP16CM-valeryl; 344, FP16CM-pivalyl; 311 nM, respectively. Collectively, results of these investigations suggest that esterification of 21-OH with various anhydrides did not improve receptor binding, inhibition of NO generation and ear edema inhibition, however, serum corticosterone level and local over systemic activities (L/S) were markedly improved.

Squirrel monkey immunophilin FKBP51 is a potent inhibitor of glucocorticoid receptor binding.

Squirrel monkeys have high circulating cortisol to compensate for expression of low-affinity glucocorticoid receptors (GRs). We have demonstrated that the FK506-binding immunophilin FKBP51 is elevated in squirrel monkey lymphocytes (SML) and, in preliminary studies, have shown that squirrel monkey FKBP51 is inhibitory to GR binding. In this report, we have demonstrated that elevated FKBP51 is the unequivocal cause of glucocorticoid resistance in SML in the following ways: 1) FK506 increased GR binding in cytosol from SML in a concentration-dependent manner, an effect reproduced by rapamycin but not cyclosporin A. The apparent K6 (6.1 nM) and rank-order of steroid displacement of [3H]dexamethasone binding in FK506-treated SML cytosol are characteristic of high-affinity GR binding. 2) cytosol from COS-7 cells expressing squirrel monkey FKBP51 inhibited GR binding in cytosol from human lymphocytes by 74%. Cytosol from COS-7 cells expressing human FKBP51 inhibited GR binding by 23%. 3) expression of squirrel monkey FKBP51 increased the median effective concentration (EC50) for dexamethasone in GR transactivation studies in COS-7 cells by approximately 17-fold, compared with the EC50 in control cells. The expression of human FKBP51 increased the EC50 for dexamethasone in COS-7 cells by less than 3-fold, compared with control. Squirrel monkey FKBP51 shares 94% overall amino acid homology with human FKBP51, with 92% and 99% homology with human FKBP51 in the peptidyl-prolyl isomerase and the tetratricopeptide repeat domains, respectively. Amino acid differences in the more variable N- or C-terminal regions or in regions which join the highly homologous functional domains may be responsible for its more potent inhibitory activity.

Glucocorticoid receptors in the euryhaline teleost Anguilla anguilla

To determine the importance of glucocorticoids in the salt water adaptation of European yellow eel we have evaluated the concentration, affinity and physical properties of glucocorticoid receptors (GR) in gill from both sea water- (SW) and freshwater-adapted (FW) animals. Using ligand binding techniques we demonstrated that high affinity GR were present in both cytosolic and nuclear fractions obtained from whole gill. Isoelectric focusing (IEF) of branchial GR indicated the presence of two distinct species, with pI values of 6.1 and 6.7. The form at pI 6.7 sedimented with a Svedberg constant of 4S on glycerol density gradients while the pI 6.1 sedimented in fractions corresponding to 9S. Treatment of the pI 6.1 form with urea (4 M) resulted in generation of the form with pI 6.7. The evidence thus suggested that the oligomeric urea-sensitive form (pI 6.1) contained a form of GR which, as a monomer, focused at pI 6.7. IEF revealed the same concentrations of the pI 6.7 form in both SW and FW. However, there was significantly more (3-fold) pI 6.1 isoform in FW than in SW, and this form decreased gradually during the course of seawater transfer. A transient increase of the nuclear-bound GR was also observed during SW adaptation. The balance between these forms could represent a dynamic parameter with important implications regarding GR function and gill responses to cortisol in salt water adaptation in teleosts.

Hormone activation induces nucleosome positioning in vivo.

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the transition in chromatin structure following hormone activation. This revealed two novel findings: hormone activation led to the establishment of specific translational positioning of nucleosomes despite the lack of significant positioning in the inactive state; and, in the active promoter, a subnucleosomal particle encompassing the glucocorticoid receptor (GR)-binding region was detected. The presence of only a single GR-binding site was sufficient for the structural transition to occur. Both basal promoter elements and ongoing transcription were dispensable. These data reveal a stepwise process in the transcriptional activation by glucocorticoid hormone.

Differential hepatic lobar gene expression in offspring exposed to altered maternal dietary protein intake.

Increased plasma fibrinogen concentrations are a recognized risk factor for coronary heart disease, and increased fibrinogen levels in adults are associated with parameters of reduced early growth. We studied fibrinogen gene expression in adult offspring of dams fed either a 20% (control) or an 8% protein diet [maternal low-protein (MLP) rats] during pregnancy and lactation and determined whether any effects were consistent between left and right liver lobes, since the fetal liver has a unique blood supply that produces differential stimuli to the left and right lobes. In MLP offspring, there was a reduction in all three fibrinogen mRNA copy numbers in the left liver lobe [left vs. right lobes for alpha-, beta-, and gamma-fibrinogen (x10(6) copies/ng total RNA): 8.04 vs. 23.16, P<0.001; 4.74 vs. 13.07, P<0.001; and 4.61 vs. 16.38, P = 0.007, respectively], with a parallel reduction in fibrinogen concentration in the left liver lobe (8.53+/-0.33 vs. 10.41 +/-0.65 arbitrary units, P = 0.014, left and right lobes, respectively). No such effect was observed in offspring of control dams. To investigate the underlying mechanism, glucocorticoid receptor function and mRNA levels were studied, since expression of fibrinogen genes is regulated by glucocorticoid hormones. The binding affinity of the high-affinity glucocorticoid receptor was reduced only in the left liver lobe of the MLP offspring (P = 0.02, left. vs. right), with a parallel reduction in this lobe in glucocorticoid receptor mRNA level (P = 0.006, left vs. right). In conclusion, maternal dietary protein restriction reduces fibrinogen gene expression, fibrinogen protein, and mRNA level and binding affinity of glucocorticoid receptors only in the left liver lobe of the adult offspring.

The novel progesterone receptor antagonists RTI 3021-012 and RTI 3021-022 exhibit complex glucocorticoid receptor antagonist activities: implications for the development of dissociated antiprogestins.

We have identified two novel compounds (RTI 3021-012 and RTI 3021-022) that demonstrate similar affinities for human progesterone receptor (PR) and display equivalent antiprogestenic activity. As with most antiprogestins, such as RU486, RTI 3021-012, and RTI 3021-022 also bind to the glucocorticoid receptor (GR) with high affinity. Unexpectedly, when compared with RU486, the RTI antagonists manifest significantly less GR antagonist activity. This finding indicates that, with respect to antiglucocorticoid function, receptor binding affinity is not a good predictor of biological activity. We have determined that the lack of a clear correlation between the GR binding affinity of the RTI compounds and their antagonist activity reflects the unique manner in which they modulate GR signaling. Previously, we proposed a two step "active inhibition" model to explain steroid receptor antagonism: 1) competitive inhibition of agonist binding; and 2) competition of the antagonist bound receptor with that activated by agonists for DNA response elements within target gene promoters. Accordingly, we observed that RU486, RTI 3021-012, and RTI 3021-022, when assayed for PR antagonist activity, accomplished both of these steps. Thus, all three compounds are "active antagonists" of PR function. When assayed on GR, however, RU486 alone functioned as an active antagonist. RTI 3021-012 and RTI 3021-022, on the other hand, functioned solely as "competitive antagonists" since they were capable of high affinity GR binding, but the resulting ligand receptor complex was unable to bind DNA. These results have important pharmaceutical implications supporting the use of mechanism based approaches to identify nuclear receptor modulators. Of equal importance, RTI 3021-012 and RTI 3021-022 are two new antiprogestins that may have clinical utility and are likely to be useful as research reagents with which to separate the effects of antiprogestins and antiglucocorticoids in physiological systems.

New steroidal anti-inflammatory antedrugs bind to macrophage glucocorticoid receptors and inhibit nitric oxide generation

In continuing efforts to synthesize potent, anti-inflammatory steroids devoid of systemic side effects, methyl 9α-fluoro-11β,17α,21-trihydroxy-3,20-dioxo-pregna-1,4-diene-16α-carboxylate (FP16CM) and its 21-acetate derivative (FP16CMAc) were recently synthesized and screened in animal models of inflammation. The compounds have now been assessed for high-affinity glucocorticoid receptor binding and glucocorticoid-mediated inhibition of nitric oxide (NO) generation in an in vitro RAW 264.7 macrophage cell culture system. Relative potencies for glucocorticoid receptor binding were 1, 1.7, and 2.4 for prednisolone (P) (IC 50 = 287 nM), FP16CM, and FP16CMAc, respectively. Concomitant relative potencies for inhibition of NO generation by macrophages stimulated with lipopolysaccharide were 1, 0.92 and 1.9 for P (IC 50 = 126 nM), FP16CM, and FP16CMAc, respectively. Collectively, results suggest that the novel antedrugs are active anti-inflammatory agents. The 9α-fluoro and 21-acetate substituent may contribute to enhanced topical potency, increased receptor binding affinity and inhibitory effects on NO generation. Inhibition of vasoactive NO may be one anti-inflammatory action of the steroidal antedrugs in vivo. Collectively, results suggest that these agents may be useful for topical application in allergic/inflammatory diseases.

Brain-corticosteroid hormone dialogue: slow and persistent.

1. The stress response system is shaped by genetic factors and life experiences, of which the effect of a neonatal life event is among the most persistent. Here we report studies focused on the "nature-nurture" question using rat lines genetically selected for extreme differences in dopamine phenotype as well as rats exposed as infants to the traumatic experience of maternal deprivation. 2. As key to the endocrine and behavioural adaptations occurring in these two animal models the hormone corticosterone (CORT) is considered. The stress hormone exerts slow and persistent genomic control over neuronal activity underlying the stress response system via high affinity mineralocorticoid (MR) and glucocorticoid receptors (GR). This action is exerted in a coordinate manner and involves after stress due to the rising CORT levels progressive activation of both receptor types. 3. Short periods of maternal separation (neonatal handling) trigger subsequently enhanced maternal care and sensory stimulation. However, a prolonged period (24 h) of depriving the infant of maternal care disrupts the stress hyporesponsive period (SHRP) and causes an inappropriate rise in CORT. During development exposure to CORT and to sensory stimulation has longlasting consequences for organization of the stress response system. 4. We find that these factors embodied by mother-pup interaction are critical for dopamine phenotype, CORT receptor dynamics and neuroendocrine regulation in adult life. The findings provide a conceptual framework to study dopamine-related psychopathology against a background of genetic predisposition, early life events, stress hormones and brain development.

A Domain of the even-skipped Protein Represses Transcription by Preventing TFIID Binding to a Promoter: Repression by Cooperative Blocking

We examined the mechanism by which the C-terminal 236 amino acids of the even-skipped protein (region CD) repress transcription. A fusion protein, CDGB, was created that contains region CD fused to the glucocorticoid receptor DNA binding domain. This protein repressed transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors, and repression was dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter. Repression by CDGB was prevented when the promoter DNA was preincubated with TFIID or TBP, whereas preincubation of the template DNA with CDGB prevented TFIID binding. Together, these results strongly imply that CDGB represses transcription by inhibiting TFIID binding, and further experiments suggested a mechanism by which this may occur. Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional CDGB molecules to low-affinity binding sites throughout the basal promoter. Binding to some of these low-affinity sites was shown to contribute to repression. Further experiments suggested that the full-length eve protein also represses transcription by the same mechanism. We speculate that occupancy of secondary sites within the basal promoter by CDGB or the eve protein inhibits subsequent TFIID binding to repress transcription, a mechanism we term cooperative blocking.