Antenatal dexamethasone exposure differentially affects distinct cortical neural progenitor cells and triggers long-term changes in murine cerebral architecture and behavior.

Antenatal administration of synthetic glucocorticoids (sGC) is the standard of care for women at risk for preterm labor before 34 gestational weeks. Despite their widespread use, the type of sGC used and their dose or the dosing regimens are not standardized in the United States of America or worldwide. Several studies have identified neural deficits and the increased risk for cognitive and psychiatric disease later in life for children administered sGC prenatally. However, the precise molecular and cellular targets of GC action in the developing brain remain largely undefined. In this study, we demonstrate that a single dose of glucocorticoid during mid-gestation in mice leads to enhanced proliferation in select cerebral cortical neural stem/progenitor cell populations. These alterations are mediated by dose-dependent changes in the expression of cell cycle inhibitors and in genes that promote cell cycle re-entry. This leads to changes in neuronal number and density in the cerebral cortex at birth, coupled to long-term alterations in neurite complexity in the prefrontal cortex and hippocampus in adolescents, and changes in anxiety and depressive-like behaviors in adults.

Different mechanisms account for extracellular-signal regulated kinase activation in distinct brain regions following global ischemia and reperfusion.

Oxidative stress after cerebral ischemia and reperfusion activates extracellular signal-regulated kinases (ERK) in brain. However, the mechanism of this activation has not been elucidated. We have previously reported that in an in vitro model of oxidative stress in immature cortical neuronal cultures, the inhibition of ERK phosphatase activity contributes to ERK1/2 activation and subsequent neuronal toxicity. This study examined whether ERK activation was associated with altered activity of ERK phosphatases in a rat cardiac arrest model. Rats in experimental groups were subjected to asphyxial cardiac arrest for 8 min and then resuscitated for 30 min. Significant ERK activation was detected in both cortex and hippocampus following ischemia/reperfusion by immunoblotting. ERK phosphatase activity was reversibly inhibited in cerebral cortex but not affected in hippocampus following ischemia/reperfusion. MEK1/2 was activated in both cerebral cortex and hippocampus following ischemia/reperfusion. Using a specific inhibitor of protein phosphatase 2A (PP2A), okadaic acid (OA), we have identified PP2A to be the major ERK phosphatase that is responsible for regulating ERK activation in ischemic brain tissues. Orthovanadate inhibited ERK phosphatase activity in brain tissues, suggesting that tyrosine phosphatases and dual specificity phosphatases may also contribute to the ERK phosphatase activity in brain tissues. Together, these data implicate ERK phosphatase in the regulation of ERK activation in distinct brain regions following global ischemia.

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.

Nuclear export: DNA-binding domains find a surprising partner.

Calreticulin, a calcium-binding protein of the endoplasmic reticulum, has been found to function as a nuclear export factor for a large family of nuclear receptors. Atypical nuclear export pathways may thus exist that regulate the compartmentalization and activity of a distinct set of transcription factors.

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.

Nuclear matrix targeting of steroid receptors: specific signal sequences and acceptor proteins.

The nuclear matrix provides the framework for various processes that occur within the nucleus such as transcription, replication, and splicing. As the composition of the nuclear matrix varies between different cell types, the matrix may influence cell-type-specific gene expression. A number of transcription factors have been shown to be associated with the nuclear matrix, including steroid hormone receptors that were the first transcriptional regulatory proteins localized to this compartment. In this review we highlight the most recent advances in our understanding of steroid hormone receptor targeting to the matrix. A specific nuclear matrix targeting signal (NMTS) has been identified within the glucocorticoid receptor (GR) that includes portions of its DNA-binding domain and tau2 transactivation domains. Distinct nuclear matrix acceptor proteins appear to interact within the GR NMTS and impart divergent effects on the transcriptional regulatory properties of the receptor.

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.

Role of molecular chaperones in subnuclear trafficking of glucocorticoid receptors.

The delivery of activated steroid receptors to high-affinity genomic sites must be efficient enough to account for the rapidity and selectivity of many transcriptional responses to steroid hormones. Thus, the signal transduction capacity of steroid hormone receptors will be influenced by the efficiency of receptor trafficking both between different subcellular compartments (that is, the cytoplasm and nucleus) and within a specific compartment (that is, the nucleus). Molecular chaperones, such as heat shock proteins, have long been recognized to play important roles in the management of protein folding in both stressed and nonstressed cells. In recent years, the participation of these proteins in various signal transduction pathways (for example, steroid hormone responses) has also been recognized. In this review, recent results that implicate a role for distinct heat shock proteins in subnuclear trafficking of glucocorticoid receptors are discussed. These studies also highlight the importance of mobilizing the cellular chaperone machinery for managing steroid receptor folding within the nucleus.

Hypothermia during reperfusion after asphyxial cardiac arrest improves functional recovery and selectively alters stress-induced protein expression.

This study examined whether prolonged hypothermia induced 1 hour after resuscitation from asphyxial cardiac arrest would improve neurologic outcome and alter levels of stress-related proteins in rats. Rats were resuscitated from 8 minutes of asphyxia resulting in cardiac arrest. Brain temperature was regulated after resuscitation in three groups: normothermia (36.8 degrees C x 24 hours), immediate hypothermia (33 degrees C x 24 hours, beginning immediately after resuscitation), and delayed hypothermia (33 degrees C x 24 hours, beginning 60 minutes after resuscitation). Mortality and neurobehavioral deficits were improved in immediate and delayed hypothermia rats relative to normothermia rats. Furthermore, both immediate and delayed hypothermia improved neuronal survival in the CA1 region of the hippocampus assessed at 14 days. In normothermia rats, the 70-kDa heat shock protein (Hsp70) and 40-kDa heat shock protein (Hsp40) were increased within 12 hours after resuscitation in the hippocampus. Delayed hypothermia attenuated the increase in Hsp70 levels in the hippocampus but did not affect Hsp70 induction in the cerebellum. Hippocampal expression of Hsp40 was not affected by hypothermia. These data indicate that prolonged hypothermia during later reperfusion improves neurologic outcome after experimental global ischemia and is associated with selective changes in the pattern of stress-induced protein expression.

Protracted nuclear export of glucocorticoid receptor limits its turnover and does not require the exportin 1/CRM1-directed nuclear export pathway.

Glucocorticoid receptors (GRs) are shuttling proteins, yet they preferentially accumulate within either the cytoplasmic or nuclear compartment when overall rates of nuclear import or export, respectively, are limiting. Hormone binding releases receptors from stable heteromeric complexes that restrict their interactions with soluble nuclear import factors and contribute to their cytoplasmic retention. Although hormone dissociation leads to the rapid release of GRs from chromatin, unliganded nuclear receptors are delayed in their export. We have used a chimeric GR that contains a heterologous, leucine-rich nuclear export signal sequence (NES) to assess the consequences of accelerated receptor nuclear export. Leucine-rich NESs utilize the exportin 1/CRM1-dependent nuclear export pathway, which can be blocked by leptomycin B (LMB). The fact that rapid nuclear export of the NES-GR chimera, but not the protracted export of wild-type GR, is sensitive to LMB, suggests that GR does not require the exportin 1/CRM1 pathway to exit the nucleus. Despite its more rapid export, the NES-GR chimera appears indistinguishable from wild-type GR in its transactivation activity in transiently transfected cells. However, accelerated nuclear export of the NES-GR chimera is associated with an increased rate of hormone-dependent down-regulation. The increase in NES-GR down-regulation is overcome by LMB treatment, thereby confirming the connection between receptor nuclear export and down-regulation. Given the presence of a nuclear recycling pathway for GR, the protracted rate of receptor nuclear export may increase the efficiency of biological responses to secondary hormone challenges by limiting receptor down-regulation and hormone desensitization.

Steroid receptor and molecular chaperone encounters in the nucleus.

Steroid hormone receptors interact with several different molecular chaperones. DeFranco and Csermely discuss how the molecular chaperones p23 and Hsp90 may serve to regulate the activity of the ligand-bound steroid receptors within the nucleus. The authors hypothesize that these chaperone proteins may have a proactive role in promoting recycling of receptors once they have interacted with chromatin and in allowing rebinding of ligand once the receptors have been recycled.

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.

Use of digitonin-permeabilized cells in studies of steroid receptor subnuclear trafficking.

The application of a cell permeabilization technique to the analysis of nuclear import has led to many major breakthroughs in our understanding of this trafficking pathway. Digitonin permeabilization maintains the nucleus in a state competent for faithful, signal-dependent translocation through the nuclear pore complex. This system has also been used to probe the mechanism of hormone-regulated nuclear import through the use of glucocorticoid receptors (GR) as a model substrate. In this report we provide detailed descriptions of the digitonin-permeabilized cell system for use in studies of GR nuclear import. In addition, we present several novel applications that expand the utility of this system to probe for mechanisms of nuclear protein export and subnuclear trafficking.

Inhibition of glucocorticoid receptor nucleocytoplasmic shuttling by okadaic acid requires intact cytoskeleton.

It has been shown previously that glucocorticoid receptors (GRs) that have undergone hormone-dependent translocation to the nucleus and have subsequently exited the nucleus upon hormone withdrawal are unable to recycle into the nucleus if cells are treated during hormone withdrawal with okadaic acid, a cell-permeable inhibitor of certain serine/threonine protein phosphatases. Using a green fluorescent protein (GFP) GR chimera (GFP-GR), we report here that okadaic acid inhibition of steroid-dependent receptor recycling to the nucleus is abrogated in cells treated for 1 h with colcemid to eliminate microtubule networks prior to steroid addition. After withdrawal of colcemid, normal cytoskeletal architecture is restored and okadaic acid inhibition of steroid-dependent GFP-GR nuclear recycling is restored. When okadaic acid is present during hormone withdrawal, GR that is recycled to the cytoplasm becomes complexed with hsp90 and binds steroid, but it does not undergo the normal agonist-dependent dissociation from hsp90 upon retreatment with steroid. However, when the cytoskeleton is disrupted by colcemid, the GR in okadaic acid-treated cells recycles from the cytoplasm to the nucleus in an agonist-dependent manner without dissociating from hsp90. This suggests that under physiological conditions where the cytoskeleton is intact, a dephosphorylation event is required for loss of high affinity binding to hsp90 that is required for receptor translocation through the cytoplasm to the nucleus along cytoskeletal tracts.

Regulation of steroid receptor subcellular trafficking.

Cellular responses to external signals often reflect alterations in gene expression. The activation of cell surface hormone or growth factor receptors upon the binding of appropriate ligands mobilizes signal transduction cascades that can ultimately impact the activity of defined sets of transcription factors. The interpretation of hormonal signals can also be initiated intracellularly, as is the case for steroid hormone receptors. In addition to recognizing specific hormones, steroid hormone receptors also function as transcription factors and directly transduce hormonal signals to activation or repression of unique target genes. The delivery of activated steroid receptors to high-affinity genomic sites must be efficient to account for the rapidity and selectivity of many transcriptional responses to steroid hormones. Thus, the signal transduction capacity of steroid hormone receptors will be affected by the efficiency of receptor trafficking both between different subcellular compartments (i.e., the cytoplasm and nucleus) and within a specific compartment (i.e., the nucleus). This article will highlight the recent advances in our understanding of subcellular and subnuclear trafficking of steroid receptors.

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.