Mechanism of action of Hic-5/androgen receptor activator 55, a LIM domain-containing nuclear receptor coactivator.

Hic-5/androgen receptor (AR) coactivator 55 (ARA55) is a group III LIM domain protein that functions as a nuclear receptor coactivator. In the present study, we examined the mechanism by which Hic-5/ARA55 potentiates glucocorticoid receptor (GR) transactivation in the A1-2 derivative of T47D breast cancer cells. Hic-5/ARA55 is an important component of GR-coactivator complexes in A1-2 cells because ablation of Hic-5/ARA55 expression by RNA interference-mediated silencing reduced GR transactivation. As shown by chromatin immunoprecipitation (ChIP) assays, Hic-5/ARA55 is recruited to glucocorticoid-responsive promoters of the mouse mammary tumor virus, c-fos, and p21 genes in response to glucocorticoid treatment. Results from sequential ChIPs established that Hic-5/ARA55 associates with GR-containing complexes at these promoters. We also used sequential ChIPs to examine Hic-5/ARA55 interactions with other well-characterized nuclear receptor coactivators and detected transcriptional intermediary factor 2, receptor-associated coactivator 3, cAMP response element binding protein-binding protein, and p300 within Hic-5/ARA55 complexes on the mouse mammary tumor virus promoter in hormone-treated cells. Ablation of Hic-5/ARA55 expression resulted in reduction of both transcriptional intermediary factor 2 and p300 recruitment to glucocorticoid-responsive promoters. Hic-5/ARA55 is also associated with the corepressor, nuclear receptor corepressor, on glucocorticoid-responsive promoters in cells not exposed to glucocorticoids. These results suggest that Hic-5/ARA55 is required for optimal GR-mediated gene expression possibly by providing a scaffold that organizes or stabilizes coactivator complexes at some hormone-responsive promoters.

Estrogen-mediated regulation of cholinergic expression in basal forebrain neurons requires extracellular-signal-regulated kinase activity.

Beyond the role estrogen plays in neuroendocrine feedback regulation involving hypothalamic neurons, other roles for estrogen in maintaining the function of CNS neurons remains poorly understood. Primary cultures of embryonic rat neurons together with radiometric assays were used to demonstrate how estrogen alters the cholinergic phenotype in basal forebrain by differentially regulating sodium-coupled high-affinity choline uptake and choline acetyltransferase activity. High-affinity choline uptake was significantly increased 37% in basal forebrain cholinergic neurons grown in the presence of a physiological dose of estrogen (5 nM) from 4 to 10 days in vitro whereas choline acetyltransferase activity was not significantly changed in the presence of 5 or 50 nM estrogen from 4 to 10 or 10 to 16 days in vitro. Newly-synthesized acetylcholine was significantly increased 35% following 6 days of estrogen treatment (10 days in vitro). These effects are in direct contrast to those found for nerve growth factor; that is, nerve growth factor can enhance the cholinergic phenotype through changes in choline acetyltransferase activity alone. This is most surprising given that mitogen-activated protein kinase and extracellular-signal-regulated kinase1/2, kinases also activated in the signaling pathway of nerve growth factor, were found to participate in the estrogen-mediated changes in the cholinergic phenotype. Likewise, general improvement in the viability of the cultures treated with estrogen does not account for the effects of estrogen as determined by lactate dehydrogenase release and nerve growth factor-responsiveness. These findings provide evidence that estrogen enhances the differentiated phenotype in basal forebrain cholinergic neurons through second messenger signaling in a manner distinct from nerve growth factor and independent of improved survival.

Hypothermia differentially increases extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun terminal kinase activation in the hippocampus during reperfusion after asphyxial cardiac arrest.

Mitogen-activated protein kinases are signal transduction mediators that have been implicated in cell survival and cell death. This study characterized the activation of pathways in the hippocampus during reperfusion after global cerebral ischemia, as well as the influence of a regimen of hypothermia that reduces ischemic cell death in the hippocampus. Circulatory arrest was induced in rats by 8 min of asphyxia. Relative levels of phosphorylated and total extracellular signal-regulated kinase, stress-activated protein kinase/c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were measured in the hippocampus after 6, 12 or 24h of reperfusion using immunoblotting. Asphyxia induced a progressive increase in phosphorylated extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase, but no change in phosphorylated p38 mitogen-activated protein kinase. Induction of mild hypothermia (33 degrees C) during reperfusion increased extracellular signal-regulated kinase phosphorylation and produced a smaller increase in stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation at 24h. Hypothermia did not alter extracellular signal-regulated kinase activation in rats not subjected to ischemia. Extracellular signal-regulated kinase activation was associated with an increase in phosphorylation of the mitogen-activated protein kinase kinase 1/2, and was inhibited by administration of the specific mitogen-activated protein kinase kinase 1/2 inhibitor SL327. Immunohistochemical staining showed an increase in active extracellular signal-regulated kinase in the CA1, CA2, CA3 and dentate gyrus regions of the hippocampus after ischemia and reperfusion. In contrast, active stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity was most intense in the CA3 and dentate gyrus regions. These data demonstrate that both extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase pathways are activated during the first 24h of reperfusion after global cerebral ischemia, and that hypothermia increases the activation of extracellular signal-regulated kinase relative to stress-activated protein kinase/c-Jun N-terminal kinase. Thus, an increase in extracellular signal-regulated kinase activation may be associated with improved neuronal survival after ischemic injury.

Interaction of the tau2 transcriptional activation domain of glucocorticoid receptor with a novel steroid receptor coactivator, Hic-5, which localizes to both focal adhesions and the nuclear matrix.

Hic-5 (hydrogen peroxide-inducible clone-5) is a focal adhesion protein that is involved in cellular senescence. In the present study, a yeast two-hybrid screen identified Hic-5 as a protein that interacts with a region of the glucocorticoid receptor that includes a nuclear matrix-targeting signal and the tau2 transcriptional activation domain. In transiently transfected mammalian cells, overexpression of Hic-5 potentiated the activation of reporter genes by all steroid receptors, excluding the estrogen receptor. The activity of the estrogen receptor and the thyroid hormone receptor was stimulated by Hic-5 in the presence but not in the absence of coexpressed coactivator GRIP1. In biochemical fractionations and indirect immunofluorescence assays, a fraction of endogenous Hic-5 in REF-52 cells and transiently expressed Hic-5 in Cos-1 cells was associated with the nuclear matrix. The C-terminal region of Hic-5, which contains seven zinc fingers arranged in four LIM domains, was required for interaction with focal adhesions, the nuclear matrix, steroid receptors, and the tau2 domain of glucocorticoid receptor. The N-terminal region of Hic-5 possesses a transcriptional activation domain and was essential for the coactivator activity of Hic-5. Given the coexisting cytoplasmic and nuclear distributions of Hic-5 and its role in steroid receptor-mediated transcriptional activation, it is proposed that Hic-5 might transmit signals that emanate at cell attachment sites and regulate transcription factors, such as steroid receptors.

Persistent activation of ERK contributes to glutamate-induced oxidative toxicity in a neuronal cell line and primary cortical neuron cultures.

Oxidative stress can trigger neuronal cell death and has been implicated in several chronic neurological diseases and in acute neurological injury. Oxidative toxicity can be induced by glutamate treatment in cells that lack ionotrophic glutamate receptors, such as the immortalized HT22 hippocampal cell line and immature primary cortical neurons. Previously, we found that neuroprotective effects of geldanamycin, a benzoquinone ansamycin, in HT22 cells were associated with a down-regulation of c-Raf-1, an upstream activator of the extracellular signal-regulated protein kinases (ERKs). ERK activation, although often attributed strictly to neuronal cell survival and proliferation, can also be associated with neuronal cell death that occurs in response to specific insults. In this report we show that delayed and persistent activation of ERKs is associated with glutamate-induced oxidative toxicity in HT22 cells and immature primary cortical neuron cultures. Furthermore, we find that U0126, a specific inhibitor of the ERK-activating kinase, MEK-1/2, protects both HT22 cells and immature primary cortical neuron cultures from glutamate toxicity. Glutamate-induced ERK activation requires the production of specific arachidonic acid metabolites and appears to be downstream of a burst of reactive oxygen species (ROS) accumulation characteristic of oxidative stress in HT22 cells. However, inhibition of ERK activation reduces glutamate-induced intracellular Ca(2+) accumulation. We hypothesize that the precise kinetics and duration of ERK activation may determine whether downstream targets are mobilized to enhance neuronal cell survival or ensure cellular demise.

Regulation of gonadotropin-releasing hormone gene transcription.

The GT1-7 cell line, derived from gonadotropin-releasing hormone (GnRH) neurons of the mouse hypothalamus, has provided a useful system for the analysis of GnRH gene regulation. We have used these cells to examine the mechanism of glucocorticoid repression of GnRH gene transcription. One GnRH negative glucocorticoid response element (nGRE) that contributes to glucocorticoid repression is not bound directly by the glucocorticoid receptor (GR). Rather, GR is tethered to this nGRE by virtue of its interaction with a DNA-bound POU domain transcription factor (i.e. Oct-1). DNA-dependent conformational changes in Oct-1 play a major role in recruiting GR to the distal nGRE and impacts transcriptional repression brought about by either glucocorticoids or tumor-promoting phorbol esters. GT1-7 cell-specific transcription of the mouse GnRH gene is controlled by an enhancer element that shares a high degree of sequence homology with the rat GnRH gene enhancer. As in the rat gene, Oct-1 is important for mGnRH enhancer activity. Furthermore, enhancer activity appears to be influenced by the DNA-dependent conformation adopted by bound Oct-1. Thus, the precise sequence recognized by Oct-1 appears to play a important role in both cell-specific and hormonal regulation of GnRH gene transcription.

Chromatin recycling of glucocorticoid receptors: implications for multiple roles of heat shock protein 90.

Unliganded glucocorticoid receptors (GRs) released from chromatin after hormone withdrawal remain associated with the nucleus within a novel subnuclear compartment that serves as a nuclear export staging area. We set out to examine whether unliganded nuclear receptors cycle between distinct subnuclear compartments or require cytoplasmic transit to regain hormone and chromatin-binding capacity. Hormone-withdrawn rat GrH2 hepatoma cells were permeabilized with digitonin to deplete cytoplasmic factors, and then hormone-binding and chromatin-binding properties of the recycled nuclear GRs were measured. We found that recycled nuclear GRs do not require cytosolic factors or ATP to rebind hormone. Nuclear GRs that rebind hormone in permeabilized cells target to high-affinity chromatin-binding sites at 30 C, but not 0 C, in the presence of ATP. Since geldanamycin, a heat shock protein-90 (hsp90)-binding drug, inhibits hormone binding to recycled nuclear GRs, hsp90 may be required to reassemble the receptor into a form capable of productive interactions with hormone. Geldanamycin also inhibits GR release from chromatin during hormone withdrawal, suggesting that hsp90 chaperone function may play multiple roles to facilitate chromatin recycling of GR.

The glucocorticoid receptor is tethered to DNA-bound Oct-1 at the mouse gonadotropin-releasing hormone distal negative glucocorticoid response element.

An element required for glucocorticoid repression of mouse gonadotropin-releasing hormone (GnRH) gene transcription, the distal negative glucocorticoid response element (nGRE), is not bound directly by glucocorticoid receptors (GRs) but is recognized by Oct-1 present in GT1-7 cell nuclear extracts or by Oct-1 purified from HeLa cells. Furthermore, purified full-length GRs interact directly with purified Oct-1 bound to the distal nGRE. Increasing the extent of distal nGRE match to an Oct-1 consensus site not only increases the affinity of Oct-1 binding, but also alters the conformation of DNA-bound Oct-1 and the pattern of protein DNA complexes formed in vitro with GT1-7 cell nuclear extracts. In addition, the interaction of purified GR with DNA-bound Oct-1 is altered when Oct-1 is bound to the consensus Oct-1 site. Mutation of the distal nGRE to a consensus Oct-1 site is also associated with reduced glucocorticoid repression in transfected GT1-7 cells. Furthermore, repression of GnRH gene transcription by 12-O-tetradecanoylphorbol-13-acetate, which utilizes sequences that overlap with the nGRE, is reversed by this distal nGRE mutation leading to activation of GnRH gene transcription. Thus, changes in the assembly of multi-protein complexes at the distal nGRE can influence the regulation of GnRH gene transcription.

Geldanamycin provides posttreatment protection against glutamate-induced oxidative toxicity in a mouse hippocampal cell line.

The benzoquinoid ansamycin geldanamycin interferes with many cell signaling pathways and is currently being evaluated as an anticancer agent. The main intracellular target of geldanamycin is the 90-kDa heat shock protein, hsp90. In this report we demonstrate that geldanamycin is effective at preventing glutamate-induced oxidative toxicity in the HT22 mouse hippocampal cell line, even if given 4 h after glutamate treatment. Geldanamycin prevents glutamate-induced internucleosomal DNA cleavage in the HT22 cells but does not reverse the depletion of glutathione levels brought about by glutamate treatment. Both anabolic and catabolic effects are generated by geldanamycin treatment of HT22 cells, as evidenced by the induction of hsp70 expression and degradation of c-Raf-1 protein, respectively. Thus, geldanamycin may provide an effective strategy for manipulating signaling pathways in neuronal cells that use hsp90 as they proceed through a programmed cell death pathway in response to oxidative stress.

The DNA-binding and tau2 transactivation domains of the rat glucocorticoid receptor constitute a nuclear matrix-targeting signal.

Using an ATP-depletion paradigm to augment glucocorticoid receptor (GR) binding to the nuclear matrix, we have identified a minimal segment of the receptor that constitutes a nuclear matrix targeting signal (NMTS). While previous studies implicated a role for the receptor's DNA-binding domain in nuclear matrix targeting, we show here that this domain of rat GR is necessary, but not sufficient, for matrix targeting. A minimal NMTS can be generated by linking the rat GR DNA-binding domain to either its tau2 transactivation domain in its natural context, or a heterologous transactivation domain derived from the Herpes simplex virus VP16 protein. The transactivation and nuclear matrix-targeting activities of tau2 are separable, as transactivation mutants were identified that either inhibited or had no apparent effect on matrix targeting of tau2. A functional interaction between the NMTS of rat GR and the RNA-binding nuclear matrix protein hnRNP U was revealed in cotransfection experiments in which hnRNP U overexpression was found to interfere with the transactivation activity of GR derivatives that possess nuclear matrix-binding capacity. We have therefore ascribed a novel function to a steroid hormone transactivation domain that could be an important component of the mechanism used by steroid hormone receptors to regulate genes in their native configuration within the nucleus.

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells.

Two morphologically distinct subpopulations of GT1-7 cells have been characterized and examined for their responsiveness to glucocorticoids. Type I cells have a neuronal phenotype, extending many lengthy processes, and express neuronal, but not glial, markers. Type II cells show weaker or negative immunostaining for neuronal markers and exhibit fewer processes. The effect of glucocorticoids on gonadotropin-releasing hormone (GnRH) secretion and gene expression was compared in type I and type II GT1-7 cells. For secretion studies, cells were attached to Cytodex beads and perifused with control medium or medium containing dexamethasone (dex). The high level of GnRH secreted by type I cells was slightly enhanced in the presence of dex, whereas dex rapidly and profoundly decreased the already low level of GnRH secreted by type II cells. Immunocytochemistry for GnRH showed dark reaction product in the cell bodies and processes of type I cells and little or no immunoreactivity in type II cells. Both the endogenous mouse GnRH mRNA and the transcriptional activity of a mouse GnRH promoter luciferase reporter gene plasmid were suppressed to a greater extent in type II cells than in type I. In electrophoretic mobility shift assays, there was no difference between type I and type II nuclear extracts in the pattern of protein-DNA complexes formed on two previously identified negative glucocorticoid response elements located at -237 to -201 and -184 to -150 bp of the mouse promoter. Both cell types contained glucocorticoid receptors (GR) by Western blot analysis. Cytosols from type I or type II cells were incubated with [3H]dex to obtain GR binding parameters. Binding data were consistent with a one-site model for dex binding in each case. Small differences in Kd (1.7 nM, type I; 3.1 nM, type II) or Bmax (approximately 3600 sites/cell, type I; approximately 1800 sites/cell, type II) were not likely to account for the differential sensitivity to dex treatment. In conclusion, nuclear alterations in type II cells leading to greater transcriptional susceptibility to dex, coupled with low GnRH storage levels, may be reflected in exquisite sensitivity of GnRH secretion to glucocorticoid repression. This represents the first example of a steroid hormone acting directly on GnRH-producing cells to alter GnRH secretion.

Subnuclear trafficking of glucocorticoid receptors in vitro: chromatin recycling and nuclear export.

We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

Glucocorticoid repression of the mouse gonadotropin-releasing hormone gene is mediated by promoter elements that are recognized by heteromeric complexes containing glucocorticoid receptor.

We have identified two regions of the mouse gonadotropin-releasing hormone (GnRH) promoter, one between -237 and -201 (distal element) and the other between -184 and -150 (proximal element), which are required for glucocorticoid repression in transiently transfected GT1-7 cells. These sequences show no similarity to known positive or negative glucocorticoid response elements (nGREs) and do not function when placed upstream of heterologous viral promoters. The glucocorticoid receptor (GR) does not bind directly to the distal or proximal promoter elements but may participate in glucocorticoid repression of GnRH gene transcription by virtue of its association within multiprotein complexes at these nGREs. Electrophoretic mobility shift assays with GT1-7 nuclear extract demonstrate the presence of GR-containing protein complexes on GnRH nGREs. One protein that co-occupies the distal nGRE in vitro along with GR is the POU domain transcription factor Oct-1. Thus, the tethering of GR to the GnRH distal nGRE, by virtue of a direct or indirect association with DNA-bound Oct-1, could play a role in hormone-dependent transcriptional repression of the GnRH gene. In contrast, Oct-1 does not appear to be a component of the GR-containing protein complex that is bound to the proximal nGRE.

ATP-dependent release of glucocorticoid receptors from the nuclear matrix.

Glucocorticoid receptors (GRs) have the capacity to shuttle between the nuclear and cytoplasmic compartments, sharing that trait with other steroid receptors and unrelated nuclear proteins of diverse function. Although nuclear import of steroid receptors, like that of nearly all other karyophilic proteins examined to date, requires ATP, there appear to be different energetic requirements for export of proteins, including steroid receptors, from nuclei. In an attempt to reveal which steps, if any, in the nuclear export pathway utilized by steroid receptors require ATP, we have used indirect immunofluorescence to visualize GRs within cells subjected to a reversible ATP depletion. Under conditions which lead to >95% depletion of cellular ATP levels within 90 min, GRs remain localized within nuclei and do not efflux into the cytoplasm. Under analogous conditions of ATP depletion, transfected progesterone receptors are also retained within nuclei. Importantly, GRs which accumulate within nuclei of ATP-depleted cells are distinguished from nuclear receptors in metabolically active cells by their resistance to in situ extraction with a hypotonic, detergent-containing buffer. GRs in ATP-depleted cells are not permanently trapped in this nuclear compartment, as nuclear receptors rapidly regain their capacity to be extracted upon restoration of cellular ATP, even in the absence of de novo protein synthesis. More extensive extraction of cells with high salt and detergent, coupled with DNase I digestion, established that a significant fraction of GRs in ATP-depleted cells are associated with an RNA-containing nuclear matrix. Quantitative Western blot (immunoblot) analysis confirmed the dramatic increase in GR binding to the nuclear matrix of ATP-depleted cells, while confocal microscopy revealed that GRs are bound to the matrix throughout all planes of the nucleus. ATP depletion does not lead to wholesale collapse of nuclear proteins onto the matrix, as the interaction of a subpopulation of simian virus 40 large tumor antigen with the nuclear matrix is not quantitatively altered in ATP-depleted Cos-1 cells. Nuclear GRs which are not bound to the nuclear matrix of metabolically active cells (i.e., a DNA-binding domain deletion mutant and a beta-galactosidase chimera possessing the GR nuclear localization signal sequence) are not recruited to the matrix upon depletion of cellular ATP. Thus, it appears that ATP depletion does not expose the GR to nuclear matrix interactions which are not normally encountered in cells but merely alters the dynamics of such interactions. The dynamic association of steroid receptors with the nuclear matrix may provide a mechanism which is utilized by these regulable transcription factors to facilitate their efficient scanning of the genome.

Assessment of glucocorticoid receptor-heat shock protein 90 interactions in vivo during nucleocytoplasmic trafficking.

Unliganded glucocorticoid receptors (GRs) that reside in the cytoplasm exist as heteromeric complexes comprised minimally of 90-kDa heat shock protein (hsp90) hsp70 and p56, a 56-kDa immunophilin. The binding of hsp90 to the GR occurs primarily through its carboxy-terminal, ligand-binding domain. Dissociation of GR-associated proteins accompanies hormone binding and leads to the exposure of its various functional domains. Although an association with hsp90 presumably masks the GR nuclear localization signal sequence, the recent demonstration of the coimport of GR and hsp90 into nuclei has led to the hypothesis that hsp90 facilitates GR interactions with the nuclear transport machinery. In this report we examined whether the dynamics of GR/hsp90 interactions in vivo influences its trafficking both into and out of the nucleus. GR/hsp90 complexes were stabilized in vivo by the introduction of sodium molybdate to cultured cells using a liposome-mediated delivery system. In agreement with previous in vitro studies, we found that stabilization of GR/hsp90 complexes in live cells severely restricts hormone-dependent nuclear import of GR. Constitutive nuclear import of a GR deletion derivative that does not bind hsp90 is unaffected by intracellular administration of molybdate, demonstrating that the inhibitory effects of molybdate require the coupling of the nuclear localization signal sequence to the GR ligand-binding domain. Interestingly, molybdate treatment traps both GR and progesterone receptor in the cytoplasm of cells chronically exposed to hormone, indicating that shuttling GRs and progesterone receptors can export, but not reimport into nuclei in the presence of molybdate. This result implies that the reassociation of recycled receptors with hsp90 must be an obligatory step for receptors that exit the nucleus to reacquire the capacity for nuclear import.

Glucocorticoid receptor phosphorylation in v-mos-transformed cells.

Nucleocytoplasmic shuttling of glucocorticoid receptors (GRs) is disrupted in v-mos-transformed cells leading to the redistribution of hormone-bound receptors from the nuclear to cytoplasmic compartments. We show here that GRs from v-mos-transformed cells are hyperphosphorylated on a specific peptide and maintain hormone-induced phosphorylations upon a prolonged hormone treatment that is associated with disruptions in its nucleocytoplasmic shuttling. Since similar effects on GR nucleocytoplasmic shuttling and phosphorylation were exerted upon treatment of nontransformed cells with the protein phosphatase inhibitor okadaic acid, we examined whether hyperphosphorylation of GRs in v-mos-transformed cells resulted from inhibition of receptor dephosphorylation. Protein phosphatase activity, measured using various substrates in vitro, was identical in cell-free extracts prepared from v-mos-transformed and nontransformed cells. Analysis of phosphate turnover in vivo from either the sum of all GR phosphorylation sites or from individual sites using pulse-chase analysis, did not reveal any significant difference between v-mos-transformed cells versus nontransformed cells. Thus, hyperphosphorylation of GR in v-mos-transformed cells does not appear to result from inhibition of GR dephosphorylation, but rather from stimulation of GR phosphorylation.

Selectivity of cell cycle regulation of glucocorticoid receptor function.

The restricted expression of some genes to distinct stages of the cell cycle is often brought about through alterations in the activity and/or abundance of specific transcription factors. Many cells have been shown to be unresponsive to glucocorticoid hormone action during the G2 phase of the mammalian cell cycle, suggesting that some activities of the glucocorticoid receptor (GR), a ligand-activated transcription factor, are subjected to cell cycle control. We show here that GR insensitivity in G2 is selective, affecting receptor-mediated transactivation from a simple glucocorticoid response element, but not repression from a composite glucocorticoid response element. Since glucocorticoid-dependent down-regulation of GR protein levels is also unaffected in G2, distinct activities of the receptor that participate in this homologous down-regulation must be operating as effectively in G2-synchronized cells as in asynchronous cells. Finally, the phosphorylation state of the GR is altered in G2-synchronized cells reflecting, in part, both site-specific phosphorylation and dephosphorylation events. These results suggest that, while GR may be a target for cell cycle regulated kinases and phosphatases, the resulting changes in receptor phosphorylation have an impact only on selected GR functions.

The two faces of a steroid antagonist: when an antagonist isn't.

Activation of protein kinase A potentiates the transcriptional response mediated by the glucocorticoid receptor in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs without detectable change in the phosphorylation of receptor. The transcriptional response to glucocorticoid or progestin agonists can be blocked by potent antagonists like RU 486. However, upon activation of protein kinase A, the antagonist action of RU 486 on both receptors is blunted. Indeed, RU 486 can itself activate transcription of a hormone-responsive promoter. The conditional agonist activity is observed with type II antagonists, those which recapitulate many of the early steps of ligand-dependent receptor activation, but not type I antagonists, which do not. These studies have now been extended to antimineralocorticoids. In COS-1 cells transfected with a mineralocorticoid receptor expression vector, treatment with 8-BromocAMP potentiates the response to the agonist aldosterone and elicits additional agonist activity in mineralocorticoid antagonists. A model is proposed wherein type II antagonist-receptor complexes occupy receptor binding sites on the genome. The antagonist, however, fails to promote a receptor conformation that can interact productively with a coactivator mediating the communication between receptor and the basal transcription apparatus. Activation of protein kinase A results in the recruitment or activation of a coactivator that permits recovery of receptor-mediated activation function.(ABSTRACT TRUNCATED AT 250 WORDS)