Kinetically Defined Mechanisms and Positions of Action of Two New Modulators of Glucocorticoid Receptor-regulated Gene Induction.

Most of the steps in, and many of the factors contributing to, glucocorticoid receptor (GR)-regulated gene induction are currently unknown. A competition assay, based on a validated chemical kinetic model of steroid hormone action, is now used to identify two new factors (BRD4 and negative elongation factor (NELF)-E) and to define their sites and mechanisms of action. BRD4 is a kinase involved in numerous initial steps of gene induction. Consistent with its complicated biochemistry, BRD4 is shown to alter both the maximal activity (Amax) and the steroid concentration required for half-maximal induction (EC50) of GR-mediated gene expression by acting at a minimum of three different kinetically defined steps. The action at two of these steps is dependent on BRD4 concentration, whereas the third step requires the association of BRD4 with P-TEFb. BRD4 is also found to bind to NELF-E, a component of the NELF complex. Unexpectedly, NELF-E modifies GR induction in a manner that is independent of the NELF complex. Several of the kinetically defined steps of BRD4 in this study are proposed to be related to its known biochemical actions. However, novel actions of BRD4 and of NELF-E in GR-controlled gene induction have been uncovered. The model-based competition assay is also unique in being able to order, for the first time, the sites of action of the various reaction components: GR < Cdk9 < BRD4 ≤ induced gene < NELF-E. This ability to order factor actions will assist efforts to reduce the side effects of steroid treatments.

Kinetically-defined component actions in gene repression.

Gene repression by transcription factors, and glucocorticoid receptors (GR) in particular, is a critical, but poorly understood, physiological response. Among the many unresolved questions is the difference between GR regulated induction and repression, and whether transcription cofactor action is the same in both. Because activity classifications based on changes in gene product level are mechanistically uninformative, we present a theory for gene repression in which the mechanisms of factor action are defined kinetically and are consistent for both gene repression and induction. The theory is generally applicable and amenable to predictions if the dose-response curve for gene repression is non-cooperative with a unit Hill coefficient, which is observed for GR-regulated repression of AP1LUC reporter induction by phorbol myristate acetate. The theory predicts the mechanism of GR and cofactors, and where they act with respect to each other, based on how each cofactor alters the plots of various kinetic parameters vs. cofactor. We show that the kinetically-defined mechanism of action of each of four factors (reporter gene, p160 coactivator TIF2, and two pharmaceuticals [NU6027 and phenanthroline]) is the same in GR-regulated repression and induction. What differs is the position of GR action. This insight should simplify clinical efforts to differentially modulate factor actions in gene induction vs. gene repression.

A kinase-independent activity of Cdk9 modulates glucocorticoid receptor-mediated gene induction.

A gene induction competition assay has recently uncovered new inhibitory activities of two transcriptional cofactors, NELF-A and NELF-B, in glucocorticoid-regulated transactivation. NELF-A and -B are also components of the NELF complex, which participates in RNA polymerase II pausing shortly after the initiation of gene transcription. We therefore asked if cofactors (Cdk9 and ELL) best known to affect paused polymerase could reverse the effects of NELF-A and -B. Unexpectedly, Cdk9 and ELL augmented, rather than prevented, the effects of NELF-A and -B. Furthermore, Cdk9 actions are not blocked either by Ckd9 inhibitors (DRB or flavopiridol) or by two Cdk9 mutants defective in kinase activity. The mode and site of action of NELF-A and -B mutants with an altered NELF domain are similarly affected by wild-type and kinase-dead Cdk9. We conclude that Cdk9 is a new modulator of GR action, that Ckd9 and ELL have novel activities in GR-regulated gene expression, that NELF-A and -B can act separately from the NELF complex, and that Cdk9 possesses activities that are independent of Cdk9 kinase activity. Finally, the competition assay has succeeded in ordering the site of action of several cofactors of GR transactivation. Extension of this methodology should be helpful in determining the site and mode of action of numerous additional cofactors and in reducing unwanted side effects.

PA1 protein, a new competitive decelerator acting at more than one step to impede glucocorticoid receptor-mediated transactivation.

Numerous cofactors modulate the gene regulatory activity of glucocorticoid receptors (GRs) by affecting one or more of the following three major transcriptional properties: the maximal activity of agonists (A(max)), the potency of agonists (EC(50)), and the partial agonist activity of antisteroids (PAA). Here, we report that the recently described nuclear protein, Pax2 transactivation domain interaction protein (PTIP)-associated protein 1 (PA1), is a new inhibitor of GR transactivation. PA1 suppresses A(max), increases the EC(50), and reduces the PAA of an exogenous reporter gene in a manner that is independent of associated PTIP. PA1 is fully active with, and strongly binds to, the C-terminal half of GR. PA1 reverses the effects of the coactivator TIF2 on GR-mediated gene induction but is unable to augment the actions of the corepressor SMRT. Analysis of competition assays between PA1 and TIF2 with an exogenous reporter indicates that the kinetic definition of PA1 action is a competitive decelerator at two sites upstream from where TIF2 acts. With the endogenous genes IGFBP1 and IP6K3, PA1 also represses GR induction, increases the EC(50), and decreases the PAA. ChIP and re-ChIP experiments indicate that PA1 accomplishes this inhibition of the two genes via different mechanisms as follows: PA1 appears to increase GR dissociation from and reduce GR transactivation at the IGFBP1 promoter regions but blocks GR binding to the IP6K3 promoter. We conclude that PA1 is a new competitive decelerator of GR transactivation and can act at more than one molecularly defined step in a manner that depends upon the specific gene.

A conserved protein motif is required for full modulatory activity of negative elongation factor subunits NELF-A and NELF-B in modifying glucocorticoid receptor-regulated gene induction properties.

NELF-B is a BRCA1-interacting protein and subunit (with NELF-A, -C/D, and -E) of the human negative elongation factor (NELF) complex, which participates in RNA polymerase II pausing shortly after transcription initiation, especially for synchronized gene expression. We now report new activities of NELF-B and other NELF complex subunits, which are to attenuate glucocorticoid receptor (GR)-mediated gene induction, reduce the partial agonist activity of an antagonist, and increase the EC50 of an agonist during nonsynchronized expression of exogenous and endogenous reporters. Stable knockdown of endogenous NELF-B has the opposite effects on an exogenous gene. The GR ligand-binding domain suffices for these biological responses. ChIP assays reveal that NELF-B diminishes GR recruitment to promoter regions of two endogenous genes. Using a new competition assay, NELF-A and NELF-B are each shown to act independently as competitive decelerators at two steps after the site of GR action and before or at the site of reporter gene activity. A common motif in each NELF was identified that is required for full activity of both NELF-A and NELF-B. These studies allow us to position the actions of two new modulators of GR-regulated transactivation, NELF-A and NELF-B, relative to other factors in the overall gene induction sequence.

Disruption of Ttll5/stamp gene (tubulin tyrosine ligase-like protein 5/SRC-1 and TIF2-associated modulatory protein gene) in male mice causes sperm malformation and infertility.

TTLL5/STAMP (tubulin tyrosine ligase-like family member 5) has multiple activities in cells. TTLL5 is one of 13 TTLLs, has polyglutamylation activity, augments the activity of p160 coactivators (SRC-1 and TIF2) in glucocorticoid receptor-regulated gene induction and repression, and displays steroid-independent growth activity with several cell types. To examine TTLL5/STAMP functions in whole animals, mice were prepared with an internal deletion that eliminated several activities of the Stamp gene. This mutation causes both reduced levels of STAMP mRNA and C-terminal truncation of STAMP protein. Homozygous targeted mutant (Stamp(tm/tm)) mice appear normal except for marked decreases in male fertility associated with defects in progressive sperm motility. Abnormal axonemal structures with loss of tubulin doublets occur in most Stamp(tm/tm) sperm tails in conjunction with substantial reduction in α-tubulin polyglutamylation, which closely correlates with the reduction in mutant STAMP mRNA. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of WT versus Stamp(tm/tm) males despite the levels of WT STAMP mRNA in testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to other Ttll genes or 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique, tissue-selective TTLL-mediated pathway for α-tubulin polyglutamylation that is required for sperm maturation and motility and may be relevant for male fertility.

The use of mode of action information in risk assessment: quantitative key events/dose-response framework for modeling the dose-response for key events.

The HESI RISK21 project formed the Dose-Response/Mode-of-Action Subteam to develop strategies for using all available data (in vitro, in vivo, and in silico) to advance the next-generation of chemical risk assessments. A goal of the Subteam is to enhance the existing Mode of Action/Human Relevance Framework and Key Events/Dose Response Framework (KEDRF) to make the best use of quantitative dose-response and timing information for Key Events (KEs). The resulting Quantitative Key Events/Dose-Response Framework (Q-KEDRF) provides a structured quantitative approach for systematic examination of the dose-response and timing of KEs resulting from a dose of a bioactive agent that causes a potential adverse outcome. Two concepts are described as aids to increasing the understanding of mode of action-Associative Events and Modulating Factors. These concepts are illustrated in two case studies; 1) cholinesterase inhibition by the pesticide chlorpyrifos, which illustrates the necessity of considering quantitative dose-response information when assessing the effect of a Modulating Factor, that is, enzyme polymorphisms in humans, and 2) estrogen-induced uterotrophic responses in rodents, which demonstrate how quantitative dose-response modeling for KE, the understanding of temporal relationships between KEs and a counterfactual examination of hypothesized KEs can determine whether they are Associative Events or true KEs.

Identification of location and kinetically defined mechanism of cofactors and reporter genes in the cascade of steroid-regulated transactivation.

A currently obscure area of steroid hormone action is where the component factors, including receptor and reporter gene, act. The DNA binding of factors can be precisely defined, but the location and timing of factor binding and action are usually not equivalent. These questions are addressed for several factors (e.g. glucocorticoid receptor (GR), reporter, TIF2, NCoR, NELF-A, sSMRT, and STAMP) using our recently developed competition assay. This assay reveals both the kinetically defined mechanism of factor action and where the above factors act relative to both each other and the equilibrium equivalent to the rate-limiting step, which we call the concentration limiting step (CLS). The utility of this competition assay would be greatly increased if the position of the CLS is invariant and if the factor acting at the CLS is known. Here we report that the exogenous GREtkLUC reporter acts at the CLS as an accelerator for gene induction by GRs in U2OS cells. This mechanism of reporter function at the CLS persists with different reporters, factors, receptors, and cell types. We, therefore, propose that the reporter gene always acts at the CLS during gene induction and constitutes a landmark around which one can order the actions of all other factors. Current data suggest that how and where GR and the short form of SMRT act is also constant. These results validate a novel and rational methodology for identifying distally acting factors that would be attractive targets for pharmaceutical intervention in the treatment of diseases involving GR-regulated genes.

Binding of the N-terminal region of coactivator TIF2 to the intrinsically disordered AF1 domain of the glucocorticoid receptor is accompanied by conformational reorganizations.

Control of gene transcription by glucocorticoid receptors (GRs) is important for many physiological processes. Like other steroid hormone receptors, the regulation of target genes by GR is mediated by two transactivation domains: activation function 1 (AF1) in the N-terminal domain and AF2 in the C-terminal ligand-binding domain (LBD). Full receptor activity requires both AF1 and -2 plus assorted coregulatory proteins. Crystal structures of the ligand-bound LBD have provided insight regarding how AF2 interacts with specific coactivators. However, despite its being the major activation domain of GRs, knowledge of AF1 structure/function has languished. This is mainly because of the highly disorganized structure of the GR N-terminal domain. This lack of AF1 structure is shared by all members of the steroid/nuclear receptor superfamily for which it has been examined and AF1 is thought to allow productive interactions with assorted cofactors via protein-induced changes in secondary/tertiary structures. To date, there are no reports of a classical coactivator altering the secondary/tertiary structure of the GR AF1 domain. Earlier, we reported an N-terminal fragment of the p160 coactivator TIF2, called TIF2.0, that binds the GR N-terminal domain and alters GR transcriptional activity. We therefore proposed that TIF2.0 binding to AF1 changes both its conformation and transcriptional activity. We now report that TIF2.0 interacts with the GR AF1 domain to increase the amount of α-helical structure in the complex. Furthermore, TIF2 coactivator activity is observed in the absence of the GR LBD in a manner that requires the AF1 domain. This contrasts with previous models where TIF2 receptor interaction domains binding to GR LBD somehow alter AF1 conformation. Our results establish for the first time that coactivators can modify the structure of the AF1 domain directly via the binding of a second region of the coactivator and suggest a molecular explanation for how coactivators increase the transcriptional activity of GR-agonist complexes.

A theoretical framework for gene induction and experimental comparisons.

Ligand-mediated gene induction by steroid receptors is a multistep process characterized by a dose-response curve for gene product that follows a first-order Hill equation. This behavior has classically been explained by steroid binding to receptor being the rate-limiting step. However, this predicts a constant potency of gene induction (EC(50)) for a given receptor-steroid complex, which is challenged by the findings that various cofactors/reagents can alter this parameter in a gene-specific manner. These properties put strong constraints on the mechanisms of gene induction and raise two questions: How can a first-order Hill dose-response curve (FHDC) arise from a multistep reaction sequence, and how do cofactors modify potency? Here we introduce a theoretical framework in which a sequence of steps yields an FHDC for the final product as a function of the initial agonist concentration. An exact determination of all constants is not required to describe the final FHDC. The theory predicts mechanisms for cofactor/reagent effects on gene-induction potency and maximal activity and it assigns a relative order to cofactors in the sequence of steps. The theory is supported by several observations from glucocorticoid receptor-mediated gene induction. It identifies the mechanism and matches the measured dose-response curves for different concentrations of the combination of cofactor Ubc9 and receptor. It also predicts that an FHDC cannot involve the DNA binding of preformed receptor dimers, which is validated experimentally. The theory is general and can be applied to any biochemical reaction that shows an FHDC.

Ligand binding domain mutations of the glucocorticoid receptor selectively modify the effects with, but not binding of, cofactors.

We previously reported that several point mutations in the ligand binding domain (LBD) of glucocorticoid receptors (GRs) marginally affect the binding affinity of the synthetic glucocorticoids dexamethasone (Dex) and deacylcortivazol (DAC). However, these mutations dramatically alter the efficacy (A(max)) and potency (EC(50)) of agonists, along with the partial agonist activity (PAA) of the antisteroid Dex-mesylate (DM), for gene induction and repression in a steroid-dependent manner. This was proposed to result, in part, from altered protein-protein interactions in the complex of GR with the coactivator TIF2 despite normal TIF2 binding. To explore the generality of this phenomenon, we now ask whether these mutations also affect the transactivation properties, but not binding, of other GR-bound factors. We find that an elevated concentration of GR, to probe unidentified cofactors, or of the comodulator Ubc9 does not reverse the effects of GR LBD mutations that increase the EC(50) and lower the PAA with the GREtkLUC reporter in both CV-1 and U2OS cells. This behavior is more dramatic with Ubc9 and the isolated GR LBD fused to the GAL4 DNA binding domain, despite normal binding of Ubc9 to the mutant GRs. Similar effects, albeit gene, steroid, and transcriptional property-specific, are seen with full-length GRs and three endogenous genes in U2OS cells. Thus, changes in simple steady-state binding capacities of mutant receptors for factors cannot account for the modified transcriptional properties. In all cases, the nuclear translocation of Dex- and DAC-bound wild-type and mutant receptors is the same. These results are consistent with the earlier results with TIF2 and support the hypothesis that small changes in the GR LBD can alter the activities of the bound cofactor without modifying cofactor binding. We propose that this separation of binding and the modulation of transactivation parameters occurs for a wide variety of GR-associated cofactors.

STAMP alters the growth of transformed and ovarian cancer cells.

BACKGROUND: Steroid receptors play major roles in the development, differentiation, and homeostasis of normal and malignant tissue. STAMP is a novel coregulator that not only enhances the ability of p160 coactivator family members TIF2 and SRC-1 to increase gene induction by many of the classical steroid receptors but also modulates the potency (or EC50) of agonists and the partial agonist activity of antisteroids. These modulatory activities of STAMP are not limited to gene induction but are also observed for receptor-mediated gene repression. However, a physiological role for STAMP remains unclear. METHODS: The growth rate of HEK293 cells stably transfected with STAMP plasmid and overexpressing STAMP protein is found to be decreased. We therefore asked whether different STAMP levels might also contribute to the abnormal growth rates of cancer cells. Panels of different stage human cancers were screened for altered levels of STAMP mRNA. Those cancers with the greatest apparent changes in STAMP mRNA were pursued in cultured cancer cell lines. RESULTS: Higher levels of STAMP are shown to have the physiologically relevant function of reducing the growth of HEK293 cells but, unexpectedly, in a steroid-independent manner. STAMP expression was examined in eight human cancer panels. More extensive studies of ovarian cancers suggested the presence of higher levels of STAMP mRNA. Lowering STAMP mRNA levels with siRNAs alters the proliferation of several ovarian cancer tissue culture lines in a cell line-specific manner. This cell line-specific effect of STAMP is not unique and is also seen for the conventional effects of STAMP on glucocorticoid receptor-regulated gene transactivation. CONCLUSIONS: This study indicates that a physiological function of STAMP in several settings is to modify cell growth rates in a manner that can be independent of steroid hormones. Studies with eleven tissue culture cell lines of ovarian cancer revealed a cell line-dependent effect of reduced STAMP mRNA on cell growth rates. This cell-line dependency is also seen for STAMP effects on glucocorticoid receptor-mediated transactivation. These preliminary findings suggest that further studies of STAMP in ovarian cancer may yield insight into ovarian cancer proliferation and may be useful in the development of biomarker panels.

STAMP, a novel predicted factor assisting TIF2 actions in glucocorticoid receptor-mediated induction and repression.

The coactivator TIF2 was predicted to interact with an unknown factor to modify both the relative inhibition in glucocorticoid receptor (GR)-mediated gene repression and several parameters of agonists and antisteroids in GR-regulated induction. Here, we describe the isolation and characterization of the predicted factor as a new 1,277-amino-acid endogenous protein (STAMP). STAMP associates with coactivators (TIF2 and SRC-1) and is selective for a subset of the steroid/nuclear receptors including GRs. Transfected STAMP increases the effects of TIF2 in GR-mediated repression and induction. Conversely, the levels of both induction and repression of endogenous genes are reduced when STAMP small interfering RNAs are used to lower the level of endogenous STAMP. Endogenous STAMP colocalizes with GR in intact cells and is recruited to the promoters of endogenous GR-induced and -repressed genes. We suggest that STAMP is an important new, downstream component of GR action in both gene activation and gene repression.

What goes on behind closed doors: physiological versus pharmacological steroid hormone actions.

Steroid-hormone-activated receptor proteins are among the best-understood class of factors for altering gene transcription in cells. Steroid receptors are of major importance in maintaining normal human physiology by responding to circulating concentrations of steroid in the nM range. Nonetheless, most studies of steroid receptor action have been conducted using the supra-physiological conditions of saturating concentrations (> or =100 nM) of potent synthetic steroid agonists. Here we summarize the recent developments arising from experiments using two clinically relevant conditions: subsaturating concentrations of agonist (to mimic the circulating concentrations in mammals) and saturating concentrations of antagonists (which are employed in endocrine therapies to block the actions of endogenous steroids). These studies have revealed new facets of steroid hormone action that could not be uncovered by conventional experiments with saturating concentrations of agonist steroids, such as a plethora of factors/conditions for the differential control of gene expression by physiological levels of steroid, a rational approach for examining the gene-specific variations in partial agonist activity of antisteroids, and a dissociation of steroid potency and efficacy that implies the existence of separate, and possibly novel, mechanistic steps and cofactors.

Dissecting transcriptional amplification by MYC.

Supraphysiological MYC levels are oncogenic. Originally considered a typical transcription factor recruited to E-boxes (CACGTG), another theory posits MYC a global amplifier increasing output at all active promoters. Both models rest on large-scale genome-wide "-omics'. Because the assumptions, statistical parameter and model choice dictates the '-omic' results, whether MYC is a general or specific transcription factor remains controversial. Therefore, an orthogonal series of experiments interrogated MYC's effect on the expression of synthetic reporters. Dose-dependently, MYC increased output at minimal promoters with or without an E-box. Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors made expression more MYC-responsive, effectively increasing MYC-amplifier gain. Mutations of conserved MYC-Box regions I and II impaired amplification, whereas MYC-box III mutations delivered higher reporter output indicating that MBIII limits over-amplification. Kinetic theory and experiments indicate that MYC activates at least two steps in the transcription-cycle to explain the non-linear amplification of transcription that is essential for global, supraphysiological transcription in cancer.

Ligand-selective transactivation and transrepression via the glucocorticoid receptor: role of cofactor interaction.

The mechanisms that determine ligand-selective transcriptional responses by the glucocorticoid receptor (GR) are not fully understood. Using a wide panel of GR ligands, we investigated the relationships between the potency and maximal response for transactivation via a glucocorticoid response element (GRE) and transrepression via both nuclear factor small ka, CyrillicB (NFsmall ka, CyrillicB) and activator protein-1 (AP-1) sites, relative binding affinity for the GR, as well as interaction with both coactivators and corepressors. The results showed ligand-selective differences in potency and efficacy for each promoter, as well as for a particular ligand between the three promoters. Ligand potency correlated with relative affinity for the GR for agonists and partial agonists in transactivation but not for transrepression. Maximal response was unrelated to relative affinity of ligand for GR for both transactivation and transrepression. A good and significant correlation between full length coactivator binding in two-hybrid assays and efficacy as well as potency of different receptor-steroid complexes for both transactivation and transrepression supports a major role for coactivator recruitment in determination of ligand-selective transcriptional activity. Furthermore, ligand-selective GR binding to GRIP-1, as determined by both two-hybrid and DNA pull down assays, correlated positively with ligand-selective efficacy for transactivation of both a synthetic GRE reporter with expressed GR as well as of an endogenous gene via endogenous GR. The receptor interacting domain of the corepressor SMRT exhibited strong interaction with both agonists and partial agonists, similar to the results for coactivators, suggesting a possible role for SMRT in activation of transcription. However, there was no correlation between ligand affinity for the GR and cofactor interaction. These results provide strong quantitative biochemical support for a model in which GR-mediated ligand-selective differential interaction with GRIP-1, SRC-1A, NCoR and SMRT is a major determinant of ligand-selective and promoter-specific differences in potency and efficacy, for both transactivation and transrepression.

Mutations of glucocorticoid receptor differentially affect AF2 domain activity in a steroid-selective manner to alter the potency and efficacy of gene induction and repression.

The transcriptional activity of steroid hormones is intimately associated with their structure. Deacylcortivazol (DAC) contains several features that were predicted to make it an inactive glucocorticoid. Nevertheless, gene induction and repression by complexes of glucocorticoid receptor (GR) with DAC occur with potency (lower EC 50) greater than and efficacy (maximal activity, or A max) equal to those of the very active and smaller synthetic glucocorticoid dexamethasone (Dex). Guided by a recent X-ray structure of DAC bound to the GR ligand binding domain (LBD), we now report that several point mutants in the LBD have little effect on the binding of either agonist steroid. However, these same mutations dramatically alter the A max and/or EC 50 of exogenous and endogenous genes in a manner that depends on steroid structure. In some cases, Dex is no longer a full agonist. These properties appear to result from a preferential inactivation of the AF2 activation domain in the GR LBD of Dex-bound, but not DAC-bound, receptors. The Dex-bound receptors display normal binding to, but a greatly reduced response to, the coactivator TIF2, thus indicating a defect in the transmission efficiency of GR-steroid complex information to the coactivator TIF2. In addition, all GR mutants that are active in gene induction with either Dex or DAC have greatly reduced activity in gene repression. This contrasts with the reports of GR mutations preferentially suppressing GR-mediated induction. The properties of these GR mutants in gene induction support the hypothesis that the A max and EC 50 of GR-controlled gene expression can be independently modified, indicate that the receptor can be modified to favor activity with a specific agonist steroid, and suggest that new ligands with suitable substituents may be able to affect the same LBD conformational changes and thereby broaden the therapeutic applications of glucocorticoid steroids.

Differential modulation of glucocorticoid and progesterone receptor transactivation.

The determinants of the different biological activities of progesterone receptors (PRs) vs. glucocorticoid receptors (GRs), which bind to the same DNA sequences, remain poorly understood. The mechanisms by which differential expression of a common target gene can be achieved by PR and GR include unequal agonist steroid concentrations for half maximal induction (EC50) and dissimilar amounts of residual partial agonist activity for antisteroids in addition to the more common changes in total gene induction, or Vmax. Several factors are known to alter some or all of these three parameters for GR-regulated gene induction and some (i.e., the corepressors NCoR and SMRT) modulate the EC50 and partial agonist activity for GR and PR induction of the same gene in opposite directions. The current study demonstrates that other factors known to modulate GR properties (GME, GMEB-2, Ubc9, and STAMP) can also differentially interact with PRs or alter several of the above induction parameters under otherwise identical conditions. These results support the hypothesis that the modulation of EC50, partial agonist activity, and Vmax by a given factor is not limited to one receptor in a specific cell line. Furthermore, the number of factors that unequally modulate PR and GR induction parameters is now greatly expanded, thereby increasing the possible mechanisms for differential gene regulation by PRs vs. GRs.

An Approach to Greater Specificity for Glucocorticoids.

Glucocorticoid steroids are among the most prescribed drugs each year. Nonetheless, the many undesirable side effects, and lack of selectivity, restrict their greater usage. Research to increase glucocorticoid specificity has spanned many years. These efforts have been hampered by the ability of glucocorticoids to both induce and repress gene transcription and also by the lack of success in defining any predictable properties that control glucocorticoid specificity. Correlations of transcriptional specificity have been observed with changes in steroid structure, receptor and chromatin conformation, DNA sequence for receptor binding, and associated cofactors. However, none of these studies have progressed to the point of being able to offer guidance for increased specificity. We summarize here a mathematical theory that allows a novel and quantifiable approach to increase selectivity. The theory applies to all three major actions of glucocorticoid receptors: induction by agonists, induction by antagonists, and repression by agonists. Simple graphical analysis of competition assays involving any two factors (steroid, chemical, peptide, protein, DNA, etc.) yields information (1) about the kinetically described mechanism of action for each factor at that step where the factor acts in the overall reaction sequence and (2) about the relative position of that step where each factor acts. These two pieces of information uniquely provide direction for increasing the specificity of glucocorticoid action. Consideration of all three modes of action indicate that the most promising approach for increased specificity is to vary the concentrations of those cofactors/pharmaceuticals that act closest to the observed end point. The potential for selectivity is even greater when varying cofactors/pharmaceuticals in conjunction with a select class of antagonists.

How much is enough? Modulation of dose-response curve for steroid receptor-regulated gene expression by changing concentrations of transcription factor.

The position of the dose-response curve for steroid-regulated gene expression determines how much variation in response will accompany the normal physiological changes in circulating steroid. Over the last several years, it has become clear that the concentration of steroid hormone required for half-maximal induction or repression by a given receptor-steroid complex, which is normally called the EC50, is not constant for all responsive genes. Thus, the position of the dose-response curve can change so that a single concentration of steroid produces very different percentages of maximal activity. This, in turn, allows for the differential expression of genes by a common steroid hormone concentration during development, differentiation, and homeostasis. Here we review the variety of factors that influence the EC50 and position of the dose-response curve for steroid hormone receptors, discuss what is known about the mechanisms, and highlight promising areas for future research.