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There is extensive knowledge of androgen receptor (AR) signaling in cancer cells, but less regarding androgen action in stromal cells of the tumor microenvironment. We report here the genome-wide effects of a stromal cell specific molecular adapter and AR coregulator, hydrogen peroxide-inducible gene 5 (Hic-5/TGFB1I1), on AR function in prostate myofibroblasts. Following androgen stimulation, Hic-5 rapidly translocates to the nucleus, coincident with increased phosphorylation of focal adhesion kinase. As a coregulator, Hic-5 acted to amplify or inhibit regulation of approximately 50% of AR target genes, affected androgen regulation of growth, cell adhesion, motility and invasion. These data suggest Hic-5 as a transferable adaptor between focal adhesions and the nucleus of prostate myofibroblasts, where it acts a key mediator of the specificity and sensitivity of AR signaling. We propose a model in which Hic-5 coordinates AR signaling with adhesion and extracellular matrix contacts to regulate cell behavior in the tumor microenvironment., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

Glucocorticoids are given to pregnant women at risk for premature delivery to promote lung maturation. Despite reports of detrimental effects of glucocorticoids on telencephalic neural stem/progenitor cells (NSPCs), the regional and cellular expressions of the glucocorticoid receptor (GR) in various NSPC populations in the intact brain have not been thoroughly assessed. Therefore in this study we performed a detailed analysis of GR protein expression in the developing mouse ventral and dorsal telencephalon in vivo. At embryonic day 11.5 (E11.5), the majority of Pax6-positive radial glial cells (RGCs) and Tbr2-positive intermediate progenitor cells (IPCs) expressed nuclear GR, while a small number of RGCs on the apical ventricular zone (aVZ), expressed cytoplasmic GR. However, on E13.5, the latter population of RGCs increased in size, whereas abventricular NSPCs and especially neurons of the cortical plate, expressed nuclear GR. In IPCs, GR was always nuclear. A similar expression profile was observed throughout the ventral telencephalon, hippocampus and olfactory bulb, with NSPCs of the aVZ primarily expressing cytoplasmic GR, while abventricular NSPCs and mature cells primarily expressed nuclear GR. Close to birth, nuclear GR accumulated within specific cortical areas such as layer V, the subplate and CA1 area of the hippocampus. In summary, our data show that GR protein is present in early NSPCs of the dorsal and ventral telencephalon at E11.5 and primarily occupies the nucleus. Moreover, our study suggests that the subcellular localization of the receptor may be subjected to region and neurodevelopmental stage-specific regulation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Androgen receptor (AR) signaling is essential for the initial development and progression of prostate cancer (PCa) as well as the growth and survival of castration-resistant tumors. However, AR action may be opposed by estrogen receptor beta (ERß) that responds to androgen metabolites produced in the prostate. The balance between the activity of these two receptors is not only influenced by the steroidogenic capacity of the prostatic microenvironment but also by its redox status and local paracrine signals such as transforming growth factor-beta (TGF-ß). In this review, we highlight the studies that revealed select roles for AR and ERß in distinct compartments of the prostate cancer microenvironment. We also discuss new work that identified stromal-epithelial crosstalk through TGF-ß1 signaling that drives the production of reactive oxygen species in stromal cells thereby selectively limiting the anti-tumor activity of ERß in cancer cells. Therefore, any new therapeutic approaches that seek to limit AR but enhance ERß activity in PCa, must take into account potential adaptive changes in the tumor microenvironment that utilize paracrine signals and altered redox balance to divert local androgen metabolites towards AR at the expense of ERß., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background/aims: The diagnostic criteria for critical illness-related corticoid insufficiency (CIRCI) are not well established, particularly for children. In addition to alterations in adrenal function, cellular resistance to glucocorticoid action could contribute to CIRCI due to alterations in the functioning of the intracellular receptor protein for corticosteroids, the glucocorticoid receptor (GR)., Methods: We have therefore undertaken a pilot, prospective study to assess whether cellular GR activity can be measured in peripheral blood mononuclear cells (PBMCs) from critically ill children., Results: Total and cytoplasmic, but not nuclear GR levels were significantly lower in PBMCs from critically ill children (i.e. sepsis/septic shock and traumatic brain injury) compared to healthy controls . While total cortisol concentrations did not differ between test groups, salivary and serum-free cortisol concentrations were significantly greater in both groups of children with critical illness. Cortisol-binding globulin levels were significantly lower in patients with sepsis/septic shock., Conclusions: The lower total and cytoplasmic receptor levels in critically ill children suggest that the GR-mediated response to exogenous glucocorticoid therapy may be limited. However, the nuclear transport of GR in critically ill patients suggests that residual receptors in these patients retain functionality and may be accessible to therapeutic treatments that maximize their activity., (Copyright © 2013 S. Karger AG, Basel.)

Age-related neurodegenerative diseases are associated with alterations in gene expression in affected neurons. One of the mechanisms that could account for this is altered subcellular localization of transcription factors, which has been observed in human post-mortem brains of each of the major neurodegenerative diseases, including Parkinson's disease (PD). The specific mechanisms are yet to be elucidated; however a potential mechanism involves alterations in nuclear transport. In this study, we examined the nucleocytoplasmic trafficking of select transcription factors in response to a PD-relevant oxidative injury, 6-hydroxydopamine (6OHDA). Utilizing a well-established model of ligand-regulated nucleocytoplasmic shuttling, the glucocorticoid receptor, we found that 6OHDA selectively impaired nuclear import through an oxidative mechanism without affecting nuclear export or nuclear retention. Interestingly, impaired nuclear import was selective as Nrf2 (nuclear factor E2-related factor 2) nuclear localization remained intact in 6OHDA-treated cells. Thus, oxidative stress specifically impacts the subcellular localization of some but not all transcription factors, which is consistent with observations in post-mortem PD brains. Our data further implicate a role for altered microtubule dependent trafficking in the differential effects of 6OHDA on transcription factor import. Oxidative disruption of microtubule-dependent nuclear transport may contribute to selective declines in transcriptional responses of aging or diseased dopaminergic cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Glucocorticoids (GCs) are an ubiquitous class of steroid hormones that exert a wide array of physiological effects. Traditionally, GC action has been considered to primarily involve transcriptional effects following the binding of hormone to the glucocorticoid receptor (GR) and subsequent activation or repression of target genes. However, a number of findings suggest that cellular responses following GC exposure may be mediated by transcription-independent, or "non-classical," mechanisms. We have added to this growing body of work by recently uncovering a novel GC signaling pathway that operates through plasma membrane GRs to limit gap junction intercellular signaling and limit the proliferation of neural progenitor cells (NPCs). In this review, we highlight our current state of knowledge of non-classical GR signaling, in particular as it applies to neuronal function. Using NPCs as a cellular model, we speculate on the components of this non-classical pathway and the mechanisms whereby a number of cytoplasmic and nuclear signaling events may be integrated.

In prostate cancer, the signals that drive cell proliferation change as tumors progress from castration-sensitive (androgen-dominant) to castration-resistant states. While the mechanisms underlying this change remain uncertain, characterization of common signaling components that regulate both stages of prostate cancer proliferation is important for developing effective treatment strategies. Here, we demonstrate that paxillin, a known cytoplasmic adaptor protein, regulates both androgen- and EGF-induced nuclear signaling. We show that androgen and EGF promoted MAPK-dependent phosphorylation of paxillin, resulting in nuclear translocation of paxillin. We found nuclear paxillin could then associate with androgen-stimulated androgen receptor (AR). This complex bound AR-sensitive promoters, retaining AR within the nucleus and regulating AR-mediated transcription. Nuclear paxillin also complexed with ERK and ELK1, mediating c-FOS and cyclin D1 expression; this was followed by proliferation. Thus, paxillin is a liaison between extranuclear MAPK signaling and nuclear transcription in response to androgens and growth factors, making it a potential regulator of both castration-sensitive and castration-resistant prostate cancer. Accordingly, paxillin was required for normal growth of human prostate cancer cell xenografts, and its expression was elevated in human prostate cancer tissue microarrays. Paxillin is therefore a potential biomarker for prostate cancer proliferation and a possible therapeutic target for prostate cancer treatment.

The tumor microenvironment plays a critical role in supporting cancer cells particularly as they disengage from limitations on their growth and motility imposed by surrounding nonreactive stromal cells. We show here that stromal-derived androgenic precursors are metabolized by DU145 human prostate cancer (PCa) cells to generate ligands for estrogen receptor-β, which act to limit their motility through transcriptional regulation of E-cadherin. Although primary human PCa-associated fibroblasts and the human WPMY-1-reactive prostate stromal cell line maintain this inherent estrogen receptor (ER)β-dependent motility inhibitor activity, they are subverted by TGF-β1 pro-oxidant signals derived from cocultured DU145 PCa cells. Specifically, stromal-produced H(2)O(2), which requires Cox-2, acts as a second paracrine factor to inhibit ERβ activity in adjacent DU145 cells. Chromatin immunoprecipitation analysis reveals that ERβ recruitment to the E-cadherin promoter is inhibited when H(2)O(2) is present. Both neutralization of H(2)O(2) with catalase and prevention of its production by silencing Cox-2 expression in stromal cells restore the motility-suppression activity of stromal-derived ERβ ligand precursors. These data suggest that reactive stromal cells may still have a capacity to limit cancer cell motility through a local endocrine network but must be protected from pro-oxidant signals triggered by cancer cell-derived TGF-β1 to exhibit this cancer-suppressive function.

In type-2 long QT (LQT2), adult women and adolescent boys have a higher risk of lethal arrhythmias, called Torsades de pointes (TdP), compared to the opposite sex. In rabbit hearts, similar sex- and age-dependent TdP risks were attributed to higher expression levels of L-type Ca(2+) channels and Na(+)-Ca(2+) exchanger, at the base of the female epicardium. Here, the effects of oestrogen and progesterone are investigated to elucidate the mechanisms whereby I(Ca,L) density is upregulated in adult female rabbit hearts. I(Ca,L) density was measured by the whole-cell patch-clamp technique on days 0-3 in cardiomyocytes isolated from the base and apex of adult female epicardium. Peak I(Ca,L) was 28% higher at the base than apex (P < 0.01) and decreased gradually (days 0-3), becoming similar to apex myocytes, which had stable currents for 3 days. Incubation with oestrogen (E2, 0.1-1.0 nm) increased I(Ca,L) (∼2-fold) in female base but not endo-, apex or male myocytes. Progesterone (0.1-10 μm) had no effect at base myocytes. An agonist of the α- (PPT, 5 nm) but not the β- (DPN, 5 nm) subtype oestrogen receptor (ERα/ERβ) upregulated I(Ca,L) like E2. Western blots detected similar levels of ERα and ERβ in male and female hearts at the base and apex. E2 increased Cav1.2α (immunocytochemistry) and mRNA (RT-PCR) levels but did not change I(Ca,L) kinetics. I(Ca,L) upregulation by E2 was suppressed by the ER antagonist ICI 182,780 (10 μm) or by inhibition of transcription (actinomycin D, 4 μm) or protein biosynthesis (cycloheximide, 70 μm). Therefore, E2 upregulates I(Ca,L) by a regional genomic mechanism involving ERα which is a known determinant of sex differences in TdP risk in LQT2.

Glucocorticoids (GCs) are used to treat pregnant women at risk for preterm delivery; however, prenatal exposure to GCs may trigger adverse neurological side effects due to reduced neural progenitor cell (NPC) proliferation. Whereas many established cell-cycle regulators impact NPC proliferation, other signaling molecules, such as the gap junction protein connexin-43 (Cx43), also influence proliferation. Gap junction intercellular communication (GJIC) is influenced by GCs in some cells, but such hormone effects have not been examined in coupled stem cells. We found that both continuous and transient exposure of embryonic day 14.5 mouse neurosphere cultures to dexamethasone (DEX) limits proliferation of coupled NPCs, which is manifested by both a reduction in S-phase progression and enhanced cell-cycle exit. A short (i.e., 1-h) DEX treatment also reduced GJIC as measured by live-cell fluorescence recovery after photobleaching, and altered the synchrony of spontaneous calcium transients in coupled NPCs. GC effects on GJIC in NPCs are transcription-independent and mediated through plasma membrane glucocorticoid receptors (GRs). This nongenomic pathway operates through lipid raft-associated GRs via a site-specific, MAPK-dependent phosphorylation of Cx43, which is linked to GR via caveolin-1 (Cav-1) and c-src. Cav-1 is essential for this nongenomic action of GR, as DEX effects on GJIC, Cx43 phosphorylation, and MAPK activation are not observed in Cav-1 knockout NPCs. As transient pharmacologic inhibition of GJIC triggers reduced S-phase progression but not enhanced cell-cycle exit, the nongenomic GR signaling pathway may operate via distinct downstream effectors to alter the proliferative capacity of NPCs.

The decision to remove or refold oxidized, denatured, or misfolded proteins by heat shock protein 70 and its binding partners is critical to determine cell fate under pathophysiological conditions. Overexpression of the ubiquitin ligase C-terminus of HSC70 interacting protein (CHIP) can compensate for failure of other ubiquitin ligases and enhance protein turnover and survival under chronic neurological stress. The ability of CHIP to alter cell fate after acute neurological injury has not been assessed. Using postmortem human tissue samples, we provide the first evidence that cortical CHIP expression is increased after ischemic stroke. Oxygen glucose deprivation in vitro led to rapid protein oxidation, antioxidant depletion, proteasome dysfunction, and a significant increase in CHIP expression. To determine if CHIP upregulation enhances neural survival, we overexpressed CHIP in vitro and evaluated cell fate 24 h after acute oxidative stress. Surprisingly, CHIP overexpressing cells fared worse against oxidative injury, accumulated more ubiquitinated and oxidized proteins, and experienced decreased proteasome activity. Conversely, using small interfering RNA to decrease CHIP expression in primary neuronal cultures improved survival after oxidative stress, suggesting that increases in CHIP observed after stroke like injuries are likely correlated with diminished survival and may negatively impact the neuroprotective potential of heat shock protein 70.

Although transcriptional effects of androgens have been extensively studied, mechanisms regulating transcription-independent (nongenomic) androgen actions are poorly understood. Previously, we have shown that paxillin, a multidomain adaptor protein, is a critical regulator of testosterone-induced MAPK-signaling during Xenopus oocyte maturation. Here we examine the nongenomic effects of dihydrotestosterone (DHT) in prostate cancer cells, focusing on how paxillin mediates Erk signaling and downstream physiologic actions. We show that in LnCAP cells DHT functions as a growth factor that indirectly activates the EGF-receptor (EGFR) via androgen receptor binding and matrix metalloproteinase-mediated release of EGFR ligands. Interestingly, siRNA-mediated knockdown of paxillin expression in androgen-dependent LnCAP cells as well as in androgen-independent PC3 cells abrogates DHT- and/or EGF-induced Erk signaling. Furthermore, EGFR-induced Erk activation requires Src-mediated phosphorylation of paxillin on tyrosines 31/118. In contrast, paxillin is not required for PKC-induced Erk signaling. However, Erk-mediated phosphorylation of paxillin on serines 83/126/130 is still needed for both EGFR and PKC-mediated cellular proliferation. Thus, paxillin serves as a specific upstream regulator of Erk in response to receptor-tyrosine kinase signaling but as a general regulator of downstream Erk actions regardless of agonist. Importantly, Erk-mediated serine phosphorylation of paxillin is also required for DHT-induced prostate-specific antigen mRNA expression in LnCAP cells as well as EGF-induced cyclin D1 mRNA expression in PC3 cells, suggesting that paxillin may regulate prostate cancer proliferation by serving as a liaison between extra-nuclear kinase signaling and intra-nuclear transcriptional signals. Thus, paxillin may prove to be a novel diagnostic or therapeutic target in prostate cancer.

Hydrogen peroxide-inducible clone 5 (Hic-5) and paxillin are members of the Group III LIM domain protein family that localize to both cell nuclei and focal adhesions and link integrin-mediated signaling to the actin cytoskeleton. Prior in vitro studies have implicated paxillin in beta-amyloid-induced cell death, but little is known about the expression and function of Hic-5 and paxillin in the brain. We performed a blinded retrospective cross-sectional study of Hic-5 and paxillin expression in the hippocampi of Alzheimer disease (AD) and control subjects using immunohistochemistry and laser scanning confocal microscopy. The analysis included assessment of the expression of phosphorylated isoforms of paxillin that reflect activation of distinct signaling pathways. We found changes in the subcellular distribution of Hic-5, paxillin, and specific phosphorylated isoforms of paxillin in the AD brains. The Hic-5 and phosphorylated isoforms of paxillin colocalized with neurofibrillary tangles. Paxillin was predominantly found in reactive astrocytes in the AD hippocampi, and activated paxillin was also detected in granulovacuolar degeneration bodies in AD. These data indicate that these important scaffolding proteins that link various intracellular signaling pathways to the extracellular matrix are modified and have altered subcellular distribution in AD.

Intracellular Zn(2+) toxicity is associated with mitochondrial dysfunction. Zn(2+) depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn(2+)-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn(2+)-induced depolarization with the effects of Ca(2+) in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca(2+) caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg(2+), ADP and cyclosporine A. Zn(2+) also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn(2+)-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca(2+) and Zn(2+) in a calcein-retention assay. Consistent with the well-documented ability of Ca(2+) to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn(2+)-treated mitochondria. Considered together, our results suggest that Ca(2+) and Zn(2+) depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn(2+)-induced depolarization, and that Zn(2+) is not a particularly potent mitochondrial inhibitor.

Identification and characterization of factors regulating intracellular localization of the androgen receptor (AR) are fundamentally important because nucleocytoplasmic trafficking of AR is a critical step in AR regulation by androgen manipulation. Normally, AR is localized to the cytoplasm in the absence of androgen. Upon ligand binding, AR translocates to the nucleus, where it can modulate transcription of AR-responsive genes. The withdrawal of androgen results in the export of unliganded AR from the nucleus to the cytoplasm, where it is transcriptionally inactive. Calreticulin has been implicated as a possible nuclear export factor for AR because the two proteins form a complex. In this study, we assessed whether the cytoplasmic localization of AR requires binding to calreticulin. To test this we substituted the calreticulin binding sequence (CBS) KVFFKR (residues 579-584) with the amino acids RLAARK in AR and monitored the cellular localization of a GFP-AR fusion protein in the absence of androgen. We also determined if knockdown or knockout of calreticulin expression affected the cytoplasmic localization of the AR. We found that a mutated CBS did not affect the localization of AR and that in the absence of androgen, AR is localized to the cytoplasm regardless of its ability to interact with calreticulin. Also, a reduction in the levels or loss of calreticulin did not affect the localization of AR. These data argue that calreticulin is not required for the cytoplasmic localization of AR.

Oxidative stress induced by glutathione depletion in the mouse HT22 neuroblastoma cell line and embryonic rat immature cortical neurons causes a delayed, sustained activation of extracellular signal-regulated kinase (ERK) 1/2, which is required for cell death. This sustained activation of ERK1/2 is mediated primarily by a selective inhibition of distinct ERK1/2-directed phosphatases either by enhanced degradation (i.e., for mitogen-activated protein kinase phosphatase-1) or as shown here by reductions in enzymatic activity (i.e., for protein phosphatase type 2A). The inhibition of ERK1/2 phosphatases in HT22 cells and immature neurons subjected to glutathione depletion results from oxidative stress because phosphatase activity is restored in cells treated with the antioxidant butylated hydroxyanisole. This leads to reduced ERK1/2 activation and neuroprotection. Furthermore, an increase in free intracellular zinc that accompanies glutathione-induced oxidative stress in HT22 cells and immature neurons contributes to selective inhibition of ERK1/2 phosphatase activity and cell death. Finally, ERK1/2 also functions to maintain elevated levels of zinc. Thus, the elevation of intracellular zinc within neurons subjected to oxidative stress can trigger a robust positive feedback loop operating through activated ERK1/2 that rapidly sets into motion a zinc-dependent pathway of cell death.

Glucocorticoids and estrogens are two classes of steroid hormones that have essential but distinct physiologic functions. Estrogens also represent a risk factor for breast cancer. It has been suggested that glucocorticoids can attenuate estrogen responses, but the mechanism by which glucocorticoids inhibit estrogenic activity is unknown. In this study, we show that activation of glucocorticoid receptor (GR) by dexamethasone (DEX) induced the expression and activity of estrogen sulfotransferase (SULT1E1 or EST), an enzyme important for the metabolic deactivation of estrogens, because sulfonated estrogens fail to activate the estrogen receptor. Treatment with DEX lowered circulating estrogens, compromised uterine estrogen responses, and inhibited estrogen-dependent breast cancer growth in vitro and in a xenograft model. We further showed that the mouse and human SULT1E1 genes are transcriptional targets of GR and deletion of Sult1e1/Est in mice abolished the DEX effect on estrogen responses. These findings have revealed a novel nuclear receptor-mediated and metabolism-based mechanism of estrogen deprivation, which may have implications in therapeutic development for breast cancers. Because glucocorticoids and estrogens are widely prescribed drugs, our results also urge caution in avoiding glucocorticoid-estrogen interactions in patients.

A variety of coregulator proteins serve as partners for nuclear receptors orchestrating the molecular events required for receptor-dependent transcriptional regulation. Some coregulators directly interact with nuclear receptors and provide a platform for recruitment of other factors that provide distinct biochemical activities that influence transcriptional efficiency. Coregulators can influence chromatin structure and activity via direct modification of histone proteins or by facilitating ATP-dependent chromatin remodeling. They also have the capacity to impact multiple steps in the transcription process including initiation, elongation, and mRNA splicing. Genetic analysis in humans and animal models are revealing the important cell and tissue-type specific actions of nuclear receptor coregulators as well and their role in human physiology and disease.

Glucocorticoid action in cells is mediated by a specific receptor protein, the glucocorticoid receptor (GR). GR is a member of a superfamily of ligand-inducible transcription factors that control a variety of physiological functions; such as, metabolism, development, and reproduction. Unliganded GR is predominantly localized within the cytoplasm but rapidly and efficiently translocates to the nucleus following hormone binding. This review will focus on the intracellular signaling pathway utilized by the GR including the mechanisms that control its intracellular trafficking, hormone binding and transcriptional regulation. Many receptor-interacting proteins are involved in distinct steps in GR signal transduction, each with a unique mechanism to regulate receptor action and providing potential drug targets for the manipulation of cellular responses to glucocorticoids.

Sepsis is associated with increased expression of TNF-alpha with subsequent activation of nuclear factor-kappa B (NF-kappaB). The glucocorticoid receptor (GR) and NF-kappaB function as mutual antagonists and induction of the latter is believed to play a major role in the acquired glucocorticoid resistance that occurs in some septic patients. GR expression and function has been reported to be elevated in septic muscle suggesting a limited effect of the activated NF-kappaB on GR function in this context. In this study, the L6 myocyte cell line was used as an in vitro model for a sepsis-like condition in skeletal muscle. While short or long term treatment with TNF-alpha had no effect on GR expression, glucocorticoid-dependent downregulation of GR occurred with a kinetic profile that is accelerated relative to that observed in most cells. This downregulation was not affected by co-treatment or prior priming of L6 cells with TNF-alpha. The synthetic glucocorticoid, dexamethasone (DEX) blunted TNF-alpha-stimulated NF-kappaB activation in L6 cells. However, although effective at activating an NF-kappaB transcriptional response, TNF-alpha treatment exerted a minimal effect in myoblasts and no effect in myotubes on GR transcriptional activity. This limited impact of TNF-alpha on GR activity was not universal as TNF-alpha and DEX exerted an additive effect on the reduction in myosin heavy chain (MHC) protein expression caused by either agent alone. Thus, the selective perseverance of GR function in the presence of increased levels of glucocorticoids and TNF-alpha during sepsis or other inflammatory states may exacerbate muscle protein breakdown.

Zinc translocation from presynaptic nerve terminals to postsynaptic neurons has generally been considered the critical step leading to the accumulation of intracellular free zinc and subsequent neuronal injury. Recent evidence, however, strongly suggests that the liberation of zinc from intracellular stores upon oxidative and nitrative stimulation contributes significantly to the toxicity of this metal not only to neurons, but also to oligodendrocytes. The exact cell death signaling pathways triggered by zinc are beginning to be deciphered. In this review, we describe how the activation of 12-lipoxygenase and mitogen-activated protein kinase (MAPK) contribute to the toxicity of liberated zinc to neurons and oligodendrocytes.

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.

Growth factors and cytokines mediate communication between the epithelial and stromal compartments of the prostate. In prostate cancer (PCa), changes in the spatial arrangements of the two compartments (i.e. basement membrane invasion), DNA mutations, or cellular dedifferentiation (i.e. myofibroblasts) leads to significant changes in gene expression within both compartments. This results in altered cytokine and/or growth factor signaling in PCa. Recently, a stromal-specific androgen receptor (AR) coactivator, Hic-5/ARA55, has been identified that may play a role in regulating expression of the growth factor and/or cytokine expression in the prostate. Specifically, Hic-5/ARA55 expression influences androgen-induced keratinocyte growth factor (KGF) expression in WPMY-1 prostate stromal cells. Because Hic-5/ARA55's expression is also altered in PCa, it may play a role in the differential cellular signaling events that occur during tumor progression.

Oocyte maturation is triggered by steroids in a transcription-independent fashion that involves an unusual positive feedback loop whereby MOS (a germ cell-specific Raf) activates MEK1, which in turn activates ERK2. ERK2 then acts back on MOS to enhance its expression and amplify the kinase signaling cascade. To date, little is known regarding other factors that regulate this powerful positive feedback kinase cascade. Here we present the scaffold molecule Paxillin as a newly recognized essential regulator of meiosis in Xenopus laevis oocytes. Reduction of Paxillin expression using RNA interference or antisense oligonucleotides completely abrogates steroid-triggered meiotic resumption. Detailed signaling studies reveal that Paxillin is acting early in the kinase cascade, because it is required for accumulation of MOS protein and complete activation of downstream kinase signaling in response to steroids. Surprisingly, full Paxillin activity also requires serine phosphorylation by a kinase downstream of MOS and MEK1, possibly ERK2. Together, these data suggest that Paxillin is an important regulator of the positive feedback effects of MEK/ERK signaling on MOS protein expression. These experiments reveal a novel and critical function for Paxillin in meiosis and support the notion that Paxillin may be a general modulator of mitogen-activated protein kinase signaling.

Glucocorticoids exert their effects in target tissues predominantly through their interaction with the glucocorticoid receptor, a member of the nuclear receptor superfamily of transcription factors. Over the years many studies have linked hormone responsiveness, both in vitro and in vivo, to the levels of both glucocorticoid and glucocorticoid receptor; furthermore, an impact of glucocorticoid receptor subcellular trafficking on hormone response has been revealed. This review will focus on the molecular mechanisms responsible for the regulation of glucocorticoid receptor trafficking and expression, and will highlight work that revealed selective physiological effects of altered glucocorticoid receptor expression. The role of alterations in glucocorticoid levels and glucocorticoid receptor function in the metabolic syndrome will also be discussed.

Huntington's disease is a neurodegenerative disease caused by an expanded polyglutamine stretch within the huntingtin protein. Transfection of mutant huntingtin causes cell toxicity and depletion of CREB binding protein (CBP) or its recruitment into huntingtin aggregates. However, the role of CBP has been controversial and the relationship between polyglutamine-induced toxicity and CBP depletion has not been examined on an individual cell basis. Using a single-cell based assay, we found that, in HT22 cells or primary neurons transfected with mutant huntingtin, cell toxicity was accompanied by CBP depletion, rather than merely recruitment. Transfection with a htt exon1 construct containing uninterrupted polyglutamine or a polyglutamine region engineered to form a compact beta structure resulted in cell toxicity. CBP depletion was accompanied by histone hypo-acetylation. CBP overexpression rescued both acetylated histone levels and cell toxicity. These data suggest that CBP dysfunction and altered gene transcription contribute to mutant htt-induced neurotoxicity.

Activation of the stress axis by glucocorticoids suppresses reproductive function in many species. Here, we performed studies to determine whether these effects are mediated at the level of the hypothalamus or pituitary or both, and to dissect the underlying molecular mechanisms, using two established rodent models. Rats were treated either chronically or acutely with glucocorticoids, and circulating gonadotropins, GnRH mRNA levels, and gonadotropin subunit mRNAs levels were measured. In model I, chronic treatment for 6 days with corticosterone (CORT) was used in adult intact male rats. CORT caused a significant decrease in serum LH but not FSH secretion compared to vehicle. Whereas pituitary LHbeta and FSHbeta mRNA levels were not affected by CORT treatment, hypothalamic GnRH mRNA was significantly decreased by 35-40%. In model II, acute blockade of the estradiol (E(2))-induced gonadotropin surge by dexamethasone (DEX) was used in 28-day-old female rats. DEX treatment resulted in substantially lower serum LH and FSH concentrations compared to vehicle, although DEX had no effect on GnRH mRNA and LHbeta mRNA levels. By contrast, FSHbeta mRNA levels were about 14-fold lower in DEX-treated females. Taken together, these results indicate that suppression of gonadotropin levels by chronic elevations in glucocorticoids/stress may be accounted for in part by suppression of GnRH mRNA levels, whereas short-term glucocorticoid treatment to block the gonadotropin surge appears to involve other mechanisms including decreased FSHbeta mRNA levels.

Prostate gland development and growth requires both androgen action and epithelial-stromal communications. In fact, androgen signaling through the androgen receptor (AR) may be important in both stromal and epithelial cells of the prostate. Because interaction of AR with the coactivator, Hic-5/ARA55, results in enhanced androgen-induced transcription, we analyzed Hic-5/ARA55 expression in prostate tissue sections from normal human donors and prostate cancer patients. In each sample, Hic-5/ARA55 expression was confined to the stromal compartment of the prostate. Furthermore, a prostate stromal cell line, WPMY-1 cells, expresses Hic-5/ARA55, which is localized both at focal adhesion complexes and within the soluble cytoplasmic compartment. The ability of Hic-5/ARA55 to shuttle between the nuclear and cytoplasmic compartments was revealed on inhibition of nuclear export with leptomycin B. Small interfering RNA ablation experiments established endogenous Hic-5/ARA55 as a coactivator for both viral and endogenous cellular AR-regulated genes. Finally, the mechanism of Hic-5/ARA55 coactivator activity in WPMY-1 cells was revealed by chromatin immunoprecipitation analysis that showed its androgen-dependent recruitment to the promoter of the stromal androgen-responsive keratinocyte growth factor gene. These data provide the first demonstration of a stromal-specific AR coactivator that has an effect on an androgen-regulated growth factor that is essential for stromal/epithelial cell communication in the prostate.

Glutamate-induced oxidative toxicity is mediated by glutathione depletion in the HT22 mouse hippocampal cell line. Previous results with pharmacological agents implicated the extracellular signal-regulated kinases-1/2 (ERK1/2) in glutamate toxicity in HT22 cells and immature embryonic rat cortical neurons. In this report, we definitively establish a role for ERK1/2 in oxidative toxicity using dominant negative MEK1 expression in transiently transfected HT22 cells to block glutamate-induced cell death. In contrast, chronic activation of ERK (i.e. brought about by transfection of constitutively active ERK2 chimera) is not sufficient to trigger HT22 cell death demonstrating that ERK1/2 activation is not sufficient for toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 min and 1 h of glutamate treatment and a second peak typically emerging after 6 h. We demonstrate here that the initial phase of ERK1/2 induction is because of activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate-induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced because of depletion of intracellular cysteine pools. Thus, ERK1/2 appears to play dual roles in HT22 cells acting as part of a cellular adaptive response during the initial phases of glutamate-induced oxidative stress and contributing to toxicity during later stages of stress.

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.

In recent years, numerous nuclear receptor-interacting proteins have been identified that influence nuclear transcription through their direct modification of chromatin. Along with coactivators that possess histone acetyltransferase (HAT) or methyltransferase activity, other coactivators that lack recognizable chromatin-modifying activity have been discovered whose mechanism of action is largely unknown. The presence of multiple protein-protein interaction motifs within mechanistically undefined coactivators suggests that they function as adaptor molecules, either recruiting or stabilizing promoter-specific protein complexes. This perspective will focus on a family of nuclear receptor coactivators (i.e., group III LIM domain proteins related to paxillin) that appear to provide a scaffold to stabilize receptor interactions with chromatin-modifying coregulators.

Mild hypothermia improves survival and neurological outcome after cardiac arrest, as well as increasing activation of the extracellular-signal-regulated kinase (ERK) in hippocampus. ERK signaling is involved in neuronal growth and survival. We tested the hypothesis that the beneficial effects of hypothermia required ERK activation. ERK activation was measured by immunoblotting with phosphorylation-specific antibodies. Rats (n = 8 per group) underwent 8 min of asphyxial cardiac arrest and were resuscitated with chest compressions, ventilation, epinephrine and bicarbonate. At 30 min after resuscitation, vehicle (50% saline:50% DMSO) or the ERK kinase inhibitor U0126 (100 microg) was infused into the lateral ventricle. Cranial temperature was kept at either 33 degrees C (hypothermia) or 37 degrees C (normothermia) between 1 and 24 h. Neurological function was assessed daily for 14 days. Surviving neurons were counted in the hippocampus. A dose of 100 mug U0126 inhibited ERK bilaterally for 12 to 24 h and decreased phosphorylation of the ERK substrates ATF-2 and CREB. As in previous studies, hypothermia improved survival, neurological and histological outcome after cardiac arrest. However, survival, neurological score and histology did not differ between U0126 and vehicle-treated rats after cardiac arrest. Therefore, a dose of U0126 sufficient to inhibit biochemical markers of ERK signaling in hippocampus does not alter the beneficial effects of hypothermia induced after resuscitation in rats and did not affect recovery of normothermia-treated rats. These results suggest that hypothermia-induced improvement in outcomes does not require ERK activation.