Sangeeta Chawla

Control of Recruitment and Transcription-Activating Function of CBP Determines Gene Regulation by NMDA Receptors and L-Type Calcium Channels

Recruitment of the coactivator CBP by signal-regulated transcription factors and stimulation of CBP activity are key regulatory events in the induction of gene transcription following Ca2+ flux through ligand- and/or voltage-gated ion channels in hippocampal neurons. The mode of Ca2+ entry (L-type Ca2+ channels versus NMDA receptors) differentially controls the CBP recruitment step to CREB, providing a molecular basis for the observed Ca2+ channel type-dependent differences in gene expression. In contrast, activation of CBP is triggered irrespective of the route of Ca2+ entry, as is activation of c-Jun, that recruits CBP independently of phosphorylation at major regulatory c-Jun phosphorylation sites, serines 63 and 73. This control of CBP recruitment and activation is likely relevant to other CBP-interacting transcription factors and represents a general mechanism through which Ca2+ signals associated with electrical activity may regulate the expression of many genes.

Neuronal activity-dependent nucleocytoplasmic shuttling of HDAC4 and HDAC5.

The class II histone deacetylases, HDAC4 and HDAC5, directly bind to and repress myogenic transcription factors of the myocyte enhancer factor‐2 (MEF‐2) family thereby inhibiting skeletal myogenesis. During muscle differentiation, repression of gene transcription by MEF‐2/HDAC complexes is relieved due to calcium/calmodulin‐dependent (CaM) kinase‐induced translocation of HDAC4 and HDAC5 to the cytoplasm. MEF‐2 proteins and HDACs are also highly expressed in the nervous system and have been implicated in neuronal survival and differentiation. Here we investigated the possibility that the subcellular localization of HDACs, and thus their ability to repress target genes, is controlled by synaptic activity in neurones. We found that, in cultured hippocampal neurones, the localization of HDAC4 and HDAC5 is dynamic and signal‐regulated. Spontaneous electrical activity was sufficient for nuclear export of HDAC4 but not of HDAC5. HDAC5 translocation to the cytoplasm was induced following stimulation of calcium flux through synaptic NMDA receptors or L‐type calcium channels; glutamate bath application (stimulating synaptic and extrasynaptic NMDA receptors) antagonized nuclear export. Activity‐induced nucleocytoplasmic shuttling of both HDACs was partially blocked by the CaM kinase inhibitor KN‐62 with HDAC5 nuclear export being more sensitive to CaM kinase inhibition than that of HDAC4. Thus, the subcellular localization of HDACs in neurones is specified by neuronal activity; differences in the activation thresholds for HDAC4 and HDAC5 nuclear export provides a mechanism for input‐specific gene expression.

Mechanisms controlling gene expression by nuclear calcium signals

Nuclear calcium is an important regulator of gene expression following membrane depolarisation of electrically excitable cells. Here we describe nuclear calcium transients in hippocampal neurons following activation of calcium influx through L-type voltage-sensitive calcium channels and N-methyl-D-aspartate (NMDA) receptors, as well as following calcium release from intracellular caffeine-sensitive stores. Increases in nuclear calcium activate gene transcription by a mechanism that is distinct from gene regulation by cytoplasmic calcium signals and involves the cAMP response element (CRE) and the CRE binding protein, CREB. The nuclear calcium/calmodulin dependent (CaM) protein kinase IV, which is expressed in cultured hippocampal neurons and in the mouse pituitary cell line AtT20, may function as a mediator of nuclear calcium-induced transcription.

Activation of β-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells

This study investigates involvement of β‐catenin signalling in regulation of p‐glycoprotein (p‐gp) expression in endothelial cells derived from brain vasculature. Pharmacological interventions that enhance or that block β‐catenin signalling were applied to primary rat brain endothelial cells and to immortalized human brain endothelial cells, hCMEC/D3, nuclear translocation of β‐catenin being determined by immunocytochemistry and by western blot analysis to confirm effectiveness of the manipulations. Using the specific glycogen synthase kinase‐3 (GSK‐3) inhibitor 6‐bromoindirubin‐3′‐oxime enhanced β‐catenin and increased p‐gp expression including activating the MDR1 promoter. These increases were accompanied by increases in p‐gp‐mediated efflux capability as observed from alterations in intracellular fluorescent calcein accumulation detected by flow cytometry. Similar increases in p‐gp expression were noted with other GSK‐3 inhibitors, i.e. 1‐azakenpaullone or LiCl. Application of Wnt agonist [2‐amino‐4‐(3,4‐(methylenedioxy) benzylamino)‐6‐(3‐methoxyphenyl)pyrimidine] also enhanced β‐catenin and increased transcript and protein levels of p‐gp. By contrast, down‐regulating the pathway using Dickkopf‐1 or quercetin decreased p‐gp expression. Similar changes were observed with multidrug resistance protein 4 and breast cancer resistance protein, both known to be present at the blood–brain barrier. These results suggest that regulation of p‐gp and other multidrug efflux transporters in brain vasculature can be influenced by β‐catenin signalling.

CREB/CBP and SRE‐interacting transcriptional regulators are fast on–off switches: duration of calcium transients specifies the magnitude of transcriptional responses

Transient increases in the intracellular calcium concentration, which are associated with electrical activation of neurones, control synapse‐to‐nucleus communication. Calcium signals differ in time and space but it is unclear exactly how this translates into stimulus‐specific gene expression. Analysis of transcription induced by calcium transients with defined durations revealed that the evoked genomic responses, unlike those following neurotrophin exposure, are not all‐or‐none but graded events. The CRE‐binding protein CREB, its coactivator CREB‐binding protein (CBP), and SRE‐interacting transcriptional regulators are fast on–off switches: their activities are induced by short‐lasting calcium signals, remain active for the duration of the signal and are rapidly shut‐off after calcium concentrations have returned to basal levels. CREB is switched on by a fast, nuclear calmodulin (CaM) kinase‐dependent mechanism that mediates CREB phosphorylation on serine 133 within 30 s of calcium entry. The second calcium‐activated route to CREB involves the MAP kinase/extracellular signal‐regulated kinase (ERK1/2) cascade. This pathway can be triggered by brief, 30–60 s calcium transients. ERK1/2 activity peaks several minutes after calcium entry and can outlast the calcium transient. The shut‐off of CREB and ERK1/2 involves rapid dephosphorylation of their activator sites. These properties of transcription factors and their regulating kinases and phosphatases provide a mechanism through which the duration of calcium signals specifies the magnitude of the transcriptional response. The decoding of temporal features of calcium transients is likely to contribute to impulse‐specific gene expression.

Nuclear Ca2+ and CaM kinase IV specify hormonal- and Notch-responsiveness

Many neuronal processes require gene activation by synaptically evoked Ca2+ transients. Ca2+‐dependent signal pathways activate some transcription factors outright, but here we report that such signals also potentiate the activation of nuclear receptors by their cognate hormone, and of CBF1 by Notch, transcription factors hitherto not thought to be Ca2+‐responsive. This potentiation is occluded by histone deacetylase inhibition, indicating a mechanism involving inactivation of co‐repressors associated with these transcription factors. Synaptic activity, acting via the nuclear Ca2+‐dependent activation of CaM kinase IV, triggers the disruption of subnuclear domains containing class II histone deacetylases (HDACs) and silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), a broad‐specificity co‐repressor which represses nuclear hormone receptors and CBF1. The sequential loss of class II HDACs and SMRT from the subnuclear domains, followed by nuclear export, is associated with disruption of SMRT interaction with its target transcription factors and sensitization of these factors to their activating signal. Counterbalancing these changes, protein phosphatase 1 promotes nuclear localization of SMRT and inactivation of nuclear receptors and CBF1. Thus, the synaptically controlled kinase‐phosphatase balance of the neuron determines the efficacy of SMRT‐mediated repression and the signal‐responsiveness of a variety of transcription factors.

Calcium waves induced by hypertonic solutions in intact frog skeletal muscle fibres

1 Regenerative Ca2+ waves and oscillations indicative of calcium‐induced calcium release (CICR) activity were induced in fully polarized, fluo‐3‐loaded, intact frog skeletal muscle fibres by exposure to hypertonic Ringer solutions. 2 The calcium waves persisted in fibres exposed to EGTA‐containing solutions, during sustained depolarization of the membrane potential or following treatment with the dihydropyridine receptor (DHPR)‐blocker nifedipine. 3 The waves were blocked by the ryanodine receptor (RyR)‐specific agents ryanodine and tetracaine, and potentiated by caffeine. 4 In addition to these pharmacological properties, the amplitudes, frequency and velocity of such hypertonicity‐induced waves closely resembled those of Ca2+ waves previously described in dyspedic skeletal myocytes expressing the cardiac RyR‐2. 5 Quantitative transmission and freeze‐fracture electronmicroscopy demonstrated a reversible cell shrinkage, transverse (T)‐tubular luminal swelling and decreased T‐sarcoplasmic reticular (SR) junctional gaps in fibres maintained in and then fixed using hypertonic solutions. 6 The findings are consistent with a hypothesis in which RyR‐Ca2+ release channels can be partially liberated from their normal control by T‐tubular DHPR‐voltage sensors in hypertonic solutions, thereby permitting CICR to operate even in such fully polarized skeletal muscle fibres.

Membrane potential stabilization in amphibian skeletal muscle fibres in hypertonic solutions

This study investigated membrane transport mechanisms influencing relative changes in cell volume (V) and resting membrane potential (Em) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of Em despite cell shrinkage, which was attributable to elevation of intracellular [Cl−] above electrochemical equilibrium through Na+–Cl− and Na+−K+−2Cl− cotransporter action following exposures to extracellular hypertonicity. Fibre volumes (V) determined by confocal microscope xz‐scanning of cutaneous pectoris muscle fibres varied linearly with [1/extracellular osmolarity], showing insignificant volume corrections, in fibres studied in Cl−‐free, normal and Na+‐free Ringer solutions and in the presence of bumetanide, chlorothiazide and ouabain. The observed volume changes following increases in extracellular tonicity were compared with microelectrode measurements of steady‐state resting potentials (Em). Fibres in isotonic Cl−‐free, normal and Na+‐free Ringer solutions showed similar Em values consistent with previously reported permeability ratios PNa/PK(0.03–0.05) and PCl/PK (∼2.0) and intracellular [Na+], [K+] and [Cl−]. Increased extracellular osmolarities produced hyperpolarizing shifts in Em in fibres studied in Cl−‐free Ringer solution consistent with the Goldman‐Hodgkin‐Katz (GHK) equation. In contrast, fibres exposed to hypertonic Ringer solutions of normal ionic composition showed no such Em shifts, suggesting a Cl−‐dependent stabilization of membrane potential. This stabilization of Em was abolished by withdrawing extracellular Na+ or by the combined presence of the Na+–Cl− cotransporter (NCC) inhibitor chlorothiazide (10 μm) and the Na+−K+−2Cl− cotransporter (NKCC) inhibitor bumetanide (10 μm), or the Na+−K+‐ATPase inhibitor ouabain (1 or 10 μm) during alterations in extracellular osmolarity. Application of such agents after such increases in tonicity only produced a hyperpolarization after a time delay, as expected for passive Cl− equilibration. These findings suggest a model that implicates the NCC and/or NKCC in fluxes that maintain [Cl−]i above its electrochemical equilibrium. Such splinting of [Cl−]i in combination with the high PCl/PK of skeletal muscle stabilizes Em despite volume changes produced by extracellular hypertonicity, but at the expense of a cellular capacity for regulatory volume increases (RVIs). In situations where PCl/PK is low, the same cotransporters would instead permit RVIs but at the expense of a capacity to stabilize Em.

CREB‐dependent Nur77 induction following depolarization in PC12 cells and neurons is modulated by MEF2 transcription factors

J. Neurochem. (2010) 112, 1065–1073.

Inverse Regulation of Plasticity-related Immediate Early Genes by Calcineurin in Hippocampal Neurons

In the mammalian hippocampus, changes in the expression of immediate early genes (IEGs) is thought to contribute to long term plastic changes in neurons brought about by learning tasks and high frequency stimulation of synapses. The phosphatase calcineurin has emerged as an important negative regulator of hippocampus-dependent learning and long term potentiation. Here we investigated the possibility that the constraining action of calcineurin on hippocampal plasticity is mediated in part by regulation of gene expression through negative control of transcription factors, such as cAMP-response element (CRE)-binding protein (CREB). We assessed the effect of calcineurin inhibitors on CREB activation by neuronal activity and show that calcineurin activity is in fact required for CREB-mediated gene expression. However, inhibition of calcineurin had disparate effects on the transcriptional induction of CREB-dependent IEGs. We find that the IEG c-fos is unaffected by suppression of calcineurin activity, the plasticity-related genes Egr1/Zif268 and Egr2/Krox-20 are up-regulated, and genes encoding the orphan nuclear hormone receptors Nor1 and Nur77 are down-regulated. We further show that the up-regulation of particular IEGs is probably due to the presence of serum response elements (SREs) in their promoters, because SRE-mediated gene expression is enhanced by calcineurin blockers. Moreover, expression of the c-fos gene, which is unaffected by calcineurin inhibitors, could be down-regulated by mutating the SRE. Conversely, SRE-mediated c-fos induction in the absence of a functional CRE was enhanced by calcineurin inhibitors. Our experiments thus implicate calcineurin as a negative regulator of SRE-dependent neuronal genes.