Glucocorticoid receptor regulates accurate chromosome segregation and is associated with malignancy.

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily, which controls programs regulating cell proliferation, differentiation, and apoptosis. We have identified an unexpected role for GR in mitosis. We discovered that specifically modified GR species accumulate at the mitotic spindle during mitosis in a distribution that overlaps with Aurora kinases. We found that Aurora A was required to mediate mitosis-driven GR phosphorylation, but not recruitment of GR to the spindle. GR was necessary for mitotic progression, with increased time to complete mitosis, frequency of mitotic aberrations, and death in mitosis observed following GR knockdown. Complementation studies revealed an essential role for the GR ligand-binding domain, but no clear requirement for ligand binding in regulating chromosome segregation. The GR N-terminal domain, and specifically phosphosites S203 and S211, were not required. Reduced GR expression results in a cell cycle phenotype, with isolated cells from mouse and human subjects showing changes in chromosome content over prolonged passage. Furthermore, GR haploinsufficient mice have an increased incidence of tumor formation, and, strikingly, these tumors are further depleted for GR, implying additional GR loss as a consequence of cell transformation. We identified reduced GR expression in a panel of human liver, lung, prostate, colon, and breast cancers. We therefore reveal an unexpected role for the GR in promoting accurate chromosome segregation during mitosis, which is causally linked to tumorigenesis, making GR an authentic tumor suppressor gene.

Quantum phase transition from triangular to stripe charge order in NbSe2.

The competition between proximate electronic phases produces a complex phenomenology in strongly correlated systems. In particular, fluctuations associated with periodic charge or spin modulations, known as density waves, may lead to exotic superconductivity in several correlated materials. However, density waves have been difficult to isolate in the presence of chemical disorder, and the suspected causal link between competing density wave orders and high-temperature superconductivity is not understood. Here we used scanning tunneling microscopy to image a previously unknown unidirectional (stripe) charge-density wave (CDW) smoothly interfacing with the familiar tridirectional (triangular) CDW on the surface of the stoichiometric superconductor NbSe(2). Our low-temperature measurements rule out thermal fluctuations and point to local strain as the tuning parameter for this quantum phase transition. We use this quantum interface to resolve two longstanding debates about the anomalous spectroscopic gap and the role of Fermi surface nesting in the CDW phase of NbSe(2). Our results highlight the importance of local strain in governing phase transitions and competing phenomena, and suggest a promising direction of inquiry for resolving similarly longstanding debates in cuprate superconductors and other strongly correlated materials.

Universal features in the photoemission spectroscopy of high-temperature superconductors.

The energy gap for electronic excitations is one of the most important characteristics of the superconducting state, as it directly reflects the pairing of electrons. In the copper-oxide high-temperature superconductors (HTSCs), a strongly anisotropic energy gap, which vanishes along high-symmetry directions, is a clear manifestation of the d-wave symmetry of the pairing. There is, however, a dramatic change in the form of the gap anisotropy with reduced carrier concentration (underdoping). Although the vanishing of the gap along the diagonal to the square Cu-O bond directions is robust, the doping dependence of the large gap along the Cu-O directions suggests that its origin might be different from pairing. It is thus tempting to associate the large gap with a second-order parameter distinct from superconductivity. We use angle-resolved photoemission spectroscopy to show that the two-gap behavior and the destruction of well-defined electronic excitations are not universal features of HTSCs, and depend sensitively on how the underdoped materials are prepared. Depending on cation substitution, underdoped samples either show two-gap behavior or not. In contrast, many other characteristics of HTSCs, such as the dome-like dependence of on doping, long-lived excitations along the diagonals to the Cu-O bonds, and an energy gap at the Brillouin zone boundary that decreases monotonically with doping while persisting above (the pseudogap), are present in all samples, irrespective of whether they exhibit two-gap behavior or not. Our results imply that universal aspects of high- superconductivity are relatively insensitive to differences in the electronic states along the Cu-O bond directions.

Electronic phase diagram of high-temperature copper oxide superconductors.

In order to understand the origin of high-temperature superconductivity in copper oxides, we must understand the normal state from which it emerges. Here, we examine the evolution of the normal state electronic excitations with temperature and carrier concentration in Bi(2)Sr(2)CaCu(2)O(8+δ) using angle-resolved photoemission. In contrast to conventional superconductors, where there is a single temperature scale T(c) separating the normal from the superconducting state, the high-temperature superconductors exhibit two additional temperature scales. One is the pseudogap scale T(∗), below which electronic excitations exhibit an energy gap. The second is the coherence scale T(coh), below which sharp spectral features appear due to increased lifetime of the excitations. We find that T(∗) and T(coh) are strongly doping dependent and cross each other near optimal doping. Thus the highest superconducting T(c) emerges from an unusual normal state that is characterized by coherent excitations with an energy gap.

Tunable superconductivity and its origin at KTaO3 interfaces.

What causes Cooper pairs to form in unconventional superconductors is often elusive because experimental signatures that connect to a specific pairing mechanism are rare. Here, we observe distinct dependences of the superconducting transition temperature Tc on carrier density n2D for electron gases formed at KTaO3 (111), (001) and (110) interfaces. For the (111) interface, a remarkable linear dependence of Tc on n2D is observed over a range of nearly one order of magnitude. Further, our study of the dependence of superconductivity on gate electric fields reveals the role of the interface in mediating superconductivity. We find that the extreme sensitivity of superconductivity to crystallographic orientation can be explained by pairing via inter-orbital interactions induced by an inversion-breaking transverse optical phonon and quantum confinement. This mechanism is also consistent with the dependence of Tc on n2D. Our study may shed light on the pairing mechanism in other superconducting quantum paraelectrics.

Effects of prolactin on hypothalamic supraoptic neurones: evidence for modulation of STAT5 expression and electrical activity.

OBJECTIVES: The study investigated the role of prolactin (PRL) in modulating STAT5 and electrical activity of magnocellular neurones in the supraoptic (SO) nucleus of male rats. METHODS: Evidence of expression of STAT5 in the SO nucleus was investigated by immunocytochemical methods. Effect of blocking prolactin receptors on STAT 5 expression was investigated by Western blotting following transfection of SO neurones with a dominant negative mutant form of the PRL receptor. Prolactin-induced changes in electrical activity were investigated by extracellular recording in hypothalamic slices. RESULTS: A high proportion of SO neurones in male rats expressed immunoreactive STAT5. Levels of activated STAT5 within the SO nucleus of PRL-treated rats was reduced following transfection with a dominant negative mutant form of the PRL receptor, as compared to rats transfected with wild type PRL receptor. Electrophysiological recordings from the SO nucleus in horizontal brain slices showed that approximately 25% of neurones were responsive to PRL, with both inhibitory and excitatory effects being observed. Cells displaying PRL responses included pharmacologically-identified oxytocinergic neurones. CONCLUSIONS: Collectively, the results suggest that central PRL targets neurones of the SO nucleus, influencing both activation of the JAK-STAT signalling pathway and neuronal excitability. Whilst the functional significance of this interaction remains to be established, it might be important in co-ordinating oxytocin secretion with physiological events associated with changes in plasma PRL, or in mediating a feedback loop in the oxytocinergic regulation of lactotrophs.

Transcriptional regulation of a BMP-6 promoter by estrogen receptor alpha.

UNLABELLED: The effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a BMP-6 promoter were compared in osteoblast-like and breast cancer cells transiently transfected with ERalpha. E2 but not ICI stimulated BMP-6 reporter activity in breast cancer cells, whereas the opposite was observed in osteoblast-like cells, associated with lack of AF-2 dependence of the response, and absent intranuclear localization of ERalpha, suggesting the involvement of a distinct ERalpha-dependent response mechanism in osteoblasts. INTRODUCTION: Previous studies suggest that the tissue-selective effect of antiestrogens on bone reflects the ability of these compounds to target certain osteoblast regulatory genes. To explore this hypothesis, we examined whether antiestrogens preferentially stimulate the bone morphogenetic protein 6 (BMP-6) promoter in bone cells, and if so, whether this activity is associated with a distinct estrogen receptor (ER)alpha-dependent response mechanism to that in other cell types. MATERIALS AND METHODS: We compared the effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a 4.3-kb BMP-6 reporter construct in osteoblast-like cells (human MG63 and SaOS-2 cells and rat ROS 17/2.8 cells), human MCF-7 and T47-D breast cancer cell lines, and HepG2 hepatoma cells, after transient transfection with ERalpha, ERbeta, and mutant ER constructs. RESULTS: E2, but not ICI, stimulated BMP-6 reporter activity by approximately 100% in MCF-7, T47-D cells, and HepG2 cells when transfected with ERalpha. In contrast, in ERalpha-transfected osteoblast-like cells, an increase in reporter activity of approximately 75% was observed after treatment with ICI but not E2. The response of MG63 cells to ICI and MCF-7 cells to E2 both required ERalpha as opposed to ERbeta and the ERalpha activation function (AF)-1 activation domain. However, whereas the AF-2 domain was also required for E2 to stimulate reporter activity in MCF-7 cells, the response to ICI in MG63 cells was AF-2 independent. In further studies where we compared the intracellular distribution of ERalpha associated with these responses, E2-dependent stimulation of the BMP-6 reporter in MCF-7 cells was associated with intranuclear localization of ERalpha, whereas extranuclear localization was seen in rat osteosarcoma cells (ROS) cells treated with ICI. CONCLUSIONS: Antiestrogens selectively stimulate BMP-6 reporter activity in osteoblast-like cells through a distinct ERalpha-dependent mechanism characterized by independence of the AF-2 domain and extranuclear localization of ERalpha.

The challenge of unconventional superconductivity.

During the past few decades, several new classes of superconductors have been discovered that do not appear to be related to traditional superconductors. The source of the superconductivity of these materials is likely different from the electron-ion interactions that are at the heart of conventional superconductivity. Developing a rigorous theory for any of these classes of materials has proven to be a difficult challenge and will remain one of the major problems in physics in the decades to come.

The HSP90 molecular chaperone cycle regulates cyclical transcriptional dynamics of the glucocorticoid receptor and its coregulatory molecules CBP/p300 during ultradian ligand treatment.

Glucocorticoid (GC) hormones are secreted from the adrenal gland in a characteristic pulsatile pattern. This ultradian secretory activity exhibits remarkable plasticity, with distinct changes in response to both physiological and stressful stimuli in humans and experimental animals. It is therefore important to understand how the pattern of GC exposure regulates intracellular signaling through the GC receptor (GR). We have previously shown that each pulse of ligand initiates rapid, transient GR activation in several physiologically relevant and functionally diverse target cell types. Using chromatin immunoprecipitation assays, we detect cyclical shifts in the net equilibrium position of GR association with regulatory elements of GC-target genes and have investigated in detail the mechanism of pulsatile transcriptional regulation of the GC-induced Period 1 gene. Transient recruitment of the histone acetyl transferase complex cAMP response element-binding protein (CREB) binding protein (CBP)/p300 is found to precisely track the ultradian hormone rhythm, resulting in transient localized net changes in lysine acetylation at GC-regulatory regions after each pulse. Pulsatile changes in histone H4 acetylation and concomitant recruitment of RNA polymerase 2 precede ultradian bursts of Period 1 gene transcription. Finally, we report the crucial underlying role of the intranuclear heat shock protein 90 molecular chaperone complex in pulsatile GR regulation. Pharmacological interference of heat shock protein 90 (HSP90) with geldanamycin during the intranuclear chaperone cycle completely ablated GR's cyclical activity, cyclical cAMP response element-binding protein (CREB) binding protein (CBP)/p300 recruitment, and the associated cyclical acetylation at the promoter region. These data imply a key role for an intact nuclear chaperone cycle in cyclical transcriptional responses, regulated in time by the pattern of pulsatile hormone.

Potential use of an estrogen-glucocorticoid receptor chimera as a drug screen for tissue selective estrogenic activity.

SERMs act as ER agonists in bone despite their antagonistic properties in other tissues. As well as inhibiting bone remodelling, this effect may involve stimulation of osteoblast activity, in light of evidence from recent in vivo studies. However, progress in exploring this action has been hampered by a lack of accurate in vitro models. For example, ER antagonists are reported to stimulate reporter assays based on estrogen target genes in osteoblasts, contrary to their inhibitory effects in vivo. We examined whether evaluating global aspects of ER function provides a more accurate reflection of ER activation in osteoblasts, based on the use of morphological and/or transcriptional read-outs with green fluorescent protein (GFP)-receptor chimeras. Osteoblast-like (ROS and U2OS) and breast cancer (MCF7) cells were transfected with a human ERalpha-GFP fusion protein, and treated with ER agonists (17beta-estradiol, and dienestrol), antagonists (ICI 182,780 and ZK 164015) and SERMs (tamoxifen, raloxifene, 4-hydroxytamoxifen (4-HT) and hexestrol). We investigated cellular compartmentalisation of these constructs by fluorescence microscopy, nuclear mobility by fluorescence recovery after photobleaching (FRAP), and global activation of estrogenic transcription using a ERE-luc reporter. SERMs caused a modest increase in ERE-luc activity in osteoblast-like cells (but not in breast cells), and a reduction in nuclear mobility in breast (but not osteoblast-like) cells. These studies were then repeated using a GFP chimera where the human GR ligand binding domain (LBD) was replaced by the human ERalpha LBD (ERGR-GFP), combined with a GRE-luc reporter. Interestingly, SERMs increased both cytoplasmic to nuclear translocation of ERGR-GFP, and GRE-luc reporter activity, in osteoblast-like (but not breast) cells. Indeed, transcriptional responses to SERMs in osteoblast-like cells were considerably greater with the ERGR/GRE-luc than the ERalpha/ERE-luc system, 4-HT inducing 300 and 25% increases in reporter activity respectively. ER antagonists were entirely without effect. We conclude that evaluation of global estrogenic activity, as opposed to activation of a specific target gene, provides a more accurate read-out for osteoblast stimulation. In particular, ERGR-mediated GRE-luc activity provides a high signal response to estrogen agonists and SERMs, in a cell context dependent manner closely resembling that observed in vivo. Further studies utilising this system are justified to explore the mechanistic basis for estrogenic stimulation of osteoblast activity, and to identify newer SERMs capable of targeting this activity.

Epidermal growth factor receptor and protein kinase C signaling to ERK2: spatiotemporal regulation of ERK2 by dual specificity phosphatases.

Spatiotemporal aspects of ERK activation are stimulus-specific and dictate cellular consequences. They are dependent upon dual specificity phosphatases (DUSPs) that bind ERK via docking domains and can both inactivate and anchor ERK in cellular compartments. Using high throughput fluorescence microscopy in combination with a system where endogenous ERKs are removed and replaced with wild-type or mutated ERK2-green fluorescent protein (GFP), we show that ERK2 activation responses to epidermal growth factor (EGF) and protein kinase C (PKC) are transient and sustained, respectively. PKC-mediated ERK2 activation is associated with prolonged nuclear localization in the dephosphorylated form, whereas EGF-stimulated ERK2 activation mediates only transient nuclear accumulation. By using short inhibitory RNAs to nuclear inducible DUSP1, -2, or -4 (alone or in combination), we demonstrate that all three of these enzymes contribute to the dephosphorylation of PKC (but not EGF)-activated ERK2 in the nucleus but that they have opposing effects on localization. DUSP2 and -4 inactivate and anchor ERK2, whereas DUSP1 dephosphorylates ERK in the nucleus but allows its traffic back to the cytoplasm. Overexpression of DUSP1, -2, or -4 prevented ERK2 activation, but only DUSP2 and -4 caused ERK2-GFP nuclear accumulation or could be immunoprecipitated with ERK2. Furthermore, protein synthesis inhibition or replacement of wild-type ERK2-GFP with docking domain mutants selectively increased PKC effects on ERK activity and altered ERK2-GFP localization. These mutations also impaired the ability of ERK2-GFP to bind DUSP2 and -4. Together, our data reveal a novel, stimulus-specific, and phosphatase-specific mechanism of ERK2 regulation in the nucleus by DUSP1, -2, and -4.

Spatiotemporal regulation of ERK2 by dual specificity phosphatases.

Although many stimuli activate extracellular signal-regulated kinases 1 and 2 (ERK1/2), the kinetics and compartmentalization of ERK1/2 signals are stimulus-dependent and dictate physiological consequences. ERKs can be inactivated by dual specificity phosphatases (DUSPs), notably the MAPK phosphatases (MKPs) and atypical DUSPs, that can both dephosphorylate and scaffold ERK1/2. Using a cell imaging model (based on knockdown of endogenous ERKs and add-back of wild-type or mutated ERK2-GFP reporters), we explored possible effects of DUSPs on responses to transient or sustained ERK2 activators (epidermal growth factor and phorbol 12,13-dibutyrate, respectively). For both stimuli, a D319N mutation (which impairs DUSP binding) increased ERK2 activity and reduced nuclear accumulation. These stimuli also increased mRNA levels for eight DUSPs. In a short inhibitory RNA screen, 12 of 16 DUSPs influenced ERK2 responses. These effects were evident among nuclear inducible MKP, cytoplasmic ERK MKP, JNK/p38 MKP, and atypical DUSP subtypes and, with the exception of the nuclear inducible MKPs, were paralleled by corresponding changes in Egr-1 luciferase activation. Simultaneous removal of all JNK/p38 MKPs or nuclear inducible MKPs revealed them as positive and negative regulators of ERK2 signaling, respectively. The effects of JNK/p38 MKP short inhibitory RNAs were not dependent on protein neosynthesis but were reversed in the presence of JNK and p38 kinase inhibitors, indicating DUSP-mediated cross-talk between MAPK pathways. Overall, our data reveal that a large number of DUSPs influence ERK2 signaling. Together with the known tissue-specific expression of DUSPs and the importance of ERK1/2 in cell regulation, our data support the potential value of DUSPs as targets for drug therapy.