Calcium entry into neuronal cells through voltage or ligand-gated ion channels triggers neuronal activity-dependent gene expression critical for adaptive changes in the nervous system. Cytoplasmic calcium transients are often accompanied by an increase in the concentration of nuclear calcium, but the functional significance of such spatially distinct calcium signals is unknown. Here we show that gene expression is differentially controlled by nuclear and cytoplasmic calcium signals which enable a single second messenger to generate diverse transcriptional responses. We used nuclear microinjection of a non-diffusible calcium chelator to block increases in nuclear, but not cytoplasmic, calcium concentrations following activation of L-type voltage-gated calcium channels. We showed that increases in nuclear calcium concentration control calcium-activated gene expression mediated by the cyclic-AMP-response element (CRE), and demonstrated that the CRE-binding protein CREB can function as a nuclear calcium-responsive transcription factor. A second signalling pathway, activating transcription through the serum-response element (SRE), is triggered by a rise in cytoplasmic calcium and does not require an increase in nuclear calcium.

Recruitment of the coactivator, CREB binding protein (CBP), by signal-regulated transcription factors, such as CREB [adenosine 3', 5'-monophosphate (cAMP) response element binding protein], is critical for stimulation of gene expression. The mouse pituitary cell line AtT20 was used to show that the CBP recruitment step (CREB phosphorylation on serine-133) can be uncoupled from CREB/CBP-activated transcription. CBP was found to contain a signal-regulated transcriptional activation domain that is controlled by nuclear calcium and calcium/calmodulin-dependent (CaM) protein kinase IV and by cAMP. Cytoplasmic calcium signals that stimulate the Ras mitogen-activated protein kinase signaling cascade or expression of the activated form of Ras provided the CBP recruitment signal but did not increase CBP activity and failed to activate CREB- and CBP-mediated transcription. These results identify CBP as a signal-regulated transcriptional coactivator and define a regulatory role for nuclear calcium and cAMP in CBP-dependent gene expression.

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.

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.

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. Supplementary materialThe following supplementary material is available for this article: Table S1 Primers used in the polymerase chain reaction. This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/10.1111/j. 1471-4159.2008.05537.x. (This link will take you to the article abstract). Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Europe PMC Funders GroupAuthor Manuscript J Neurochem. Author manuscript; available in PMC 2015 January 23. et al. 2004). In this signalling pathway, interactions of Wnt proteins with the cell surface Frizzled receptors and associated membrane proteins lead to inactivation of glycogen synthase kinase-3 (GSK-3), resulting in stabilization of β-catenin. As a result, free β-catenin is allowed to accumulate and be translocated to the nucleus, binding to the transcription factor Tcf/Lef to alter the expression of target genes (Logan and Nusse 2004). Wnt proteins can also activate non-canonical pathways that do not involve β-catenin.There is evidence that Wnt signalling, particularly via the canonical pathway plays a role in vascular endothelial survival and proliferation (Wright et al. 1999;Masckauchan et al. 2005). Wnt ligands and Wnt ligand receptors have been identified in different types of vascular endothelial cells (Goodwin et al. 2006). Interactions between canonical and noncanonical pathways may be such that the one then modulates the ...

Calcium ions are the principal second messenger in the control of gene expression by electrical activation of neurons. However, the full complexity of calcium-signaling pathways leading to transcriptional activation and the cellular machinery involved are not known. Using the c-fos gene as a model system, we show here that the activity of its complex promoter is controlled by three independently operating signaling mechanisms and that their functional significance is cell type-dependent. The serum response element (SRE), which is composed of a ternary complex factor (TCF) and a serum response factor (SRF) binding site, integrates two calcium-signaling pathways. In PC12 cells, calcium-regulated transcription mediated by the SRE requires the TCF site and is not inhibited by expression of the dominant-negative Ras mutant, RasN17, nor by the MAP kinase kinase 1 inhibitor PD 98059. In contrast, TCF-dependent transcriptional regulation by nerve growth factor or epidermal growth factor is mediated by a Ras/MAP kinases (ERKs) pathway targeting the TCF Elk-1. In AtT20 cells and hippocampal neurons, calcium signals can stimulate transcription via a TCFindependent mechanism that requires the SRF binding site. The cyclic AMP response element (CRE), which cooperates with the TCF site in growth factor-regulated transcription, is a target of a third calcium-regulated pathway that is little affected by the expression of RasN17 or by PD 98059. Thus, calcium can stimulate gene expression via a TCF-, SRF-, and CRE-linked pathway that can operate independently of the Ras/MAP kinases (ERKs) signaling cascade in a cell type-dependent manner.

steady-state resting potentials (E m ). Fibres in isotonic Cl− -free, normal and Na + -free Ringer solutions showed similar E m values consistent with previously reported permeability ratios P Na /P K (0.03-0.05) and P Cl /P K (∼2.0) and intracellular [Na + ], [K + ] and [Cl − ]. Increased extracellular osmolarities produced hyperpolarizing shifts in E m 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 E m shifts,suggestingaCl − -dependentstabilizationofmembranepotential.ThisstabilizationofE m 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 P Cl /P K of skeletal muscle stabilizes E m despite volume changes produced by extracellular hypertonicity, but at the expense of a cellular capacity for regulatory volume increases (RVIs). In situations where P Cl /P K is low, the same cotransporters would instead permit RVIs but at the expense of a capacity to stabilize E m . This study investigated the relationship between changes in cell volume (V ) and resting membrane potential (E m ) in amphibian skeletal muscle fibres exposed to osmotic stress brought about by increased Emily A. Ferenczi and James A. Fraser were equal contributors to this paper. extracellular osmolarity. It was prompted by classic reports that the osmotic and electrophysiological properties of Cl − -depleted frog skeletal muscle followed predictions for a freely distensible, semipermeable sac containing a fixed quantity of solute in which cell volume decreased linearly with extracellular osmotic pressure and that this volume change resulted in membrane hyperpolarization reflecting the increased (Adrian, 1956;Blinks, 1965). Significant, osmotically induced cell volume changes in skeletal muscle similarly occur in vivo during normal activity (Sjojaard et al. 1985;Kowalchuk et al. 1998).The present experiments extended the above reports. First, they introduced an assessment of cell volume (V ) using confocal microscope scanning of amphibian skeletal muscle fibres in the xz-plane over time. This offered significant advantages over earlier measurements that only assessed steady-state fibre diameter in response to extracellular osmotic change (cf. Dydynska & Wilkie, 1963;Blinks, 1965). The cylindrical geometry of skeletal muscle fibres made it possible to simplify earlier confoca...

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