Mechanisms of Ca(2+)-dependent transcription.

Ca(2+) has a central role in coupling synaptic activity and transcriptional responses. Recent studies have focused on Ca(2+)-dependent nuclear mechanisms that bring to the nucleosomal level cascades of events initiated in the submembranous space at the synapse. In addition, a new Ca(2+)-dependent interaction between a calcium sensor and DNA has been shown to regulate transcription directly.

Interleukin 3-dependent activation of DREAM is involved in transcriptional silencing of the apoptotic Hrk gene in hematopoietic progenitor cells.

The apoptotic protein Hrk is expressed in hematopoietic progenitors after growth factor deprivation. Here we identify a silencer sequence in the 3' untranslated region of the hrk gene that binds to the transcriptional repressor DREAM in interleukin-3 (IL-3)-dependent hematopoietic progenitor cells, and abrogates the expression of reporter genes when located downstream of the open reading frame. In addition, the binding of DREAM to the hrk gene is reduced or eliminated when cells are cultured in the absence of IL-3 or treated with a calcium ionophore or a phosphatidylinositol 3-kinase-specific inhibitor, suggesting that both calcium mobilization and phosphorylation can regulate the transcriptional activity of DREAM. Furthermore, we have shown that DREAM is phosphorylated by a phosphatidylinositol 3-kinase-dependent, but Akt-independent pathway. In all cases, loss of the DREAM-DNA binding complex was correlated with increased levels of Hrk and apoptosis. These data suggest that IL-3 may trigger the activation of DREAM through different signaling pathways, which in turn binds to a silencer sequence in the hrk gene and blocks transcription, avoiding inappropriate cell death in hematopoietic progenitors.

Ca2+-dependent transcriptional repression and derepression: DREAM, a direct effector.

Control of gene expression by Ca2+ is a well known phenomenon acting through three major pathways: (i) changes in the transactivating properties of transcription factors after induction of Ca2+-dependent kinases and phosphatases (ii) Ca2+-dependent interaction between calmodulin and S-100 proteins with basic helix-loop-helix (bHLH) transcription factors that prevents binding to DNA and (iii) direct interaction between Ca2+-free DREAM and DNA that represses transcription. Because the first mechanism has been extensively reviewed, (Gallin, W. J., Greenberg, M. E. (1995). Calcium regulation of gene expression in neurons: the mode of entry matters. Curr Opin Neurobiol 5: 367-374; Santella, L., Carafoli, E. (1997). Calcium signaling in the cell nucleus. FASEB J, 11: 1091-1109) this commentary will focus on the other two with special emphasis on DREAM, the first EF-hand protein known to specifically bind DNA and regulate transcription in a Ca2+-dependent manner (Carrion, A. M.; Link, W. A., Ledo, F., Mellstrom, B., Naranjo, J. R. (1999). DREAM is a Ca2+-regulated transcriptional repressor, Nature. 398: 80-84).

The DREAM-DRE interaction: key nucleotides and dominant negative mutants.

Transcriptional repressor DREAM, an EF-hand containing calcium-binding protein, blocks basal expression of target genes through specific interaction with DRE sites in the DNA. The sequence GTCA forms the central core of the DRE site, whereas flanking nucleotides contribute notably to the affinity for DREAM. Release of binding of DREAM from the DRE results in derepression, a process that is regulated by Ca(2+). Change of two amino acids within an EF-hand in DREAM blocks Ca(2+)-induced derepression and results in potent dominant negative mutants of endogenous DREAM.

DREAM-alphaCREM interaction via leucine-charged domains derepresses downstream regulatory element-dependent transcription.

Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, alphaCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of alphaCREM is not required for the interaction, but phospho-alphaCREM shows higher affinity for DREAM. The interaction with alphaCREM is independent of the Ca(2+)-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both alphaCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca(2+).

Induction of glycerol phosphate dehydrogenase gene expression during seizure and analgesia.

Using mRNA differential display, we found that the gene for NAD(+)-dependent glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) is induced in rat brain following seizure activity. Northern blot and in situ hybridization analysis confirmed the differential display results; they also showed, in a separate model of neuronal activation, that after thermal noxious stimulation of the hind-paws, a similar increase in GPDH mRNA occurs in the areas of somatotopic projection in the lumbar spinal cord. Surprisingly, administration of analgesic doses of morphine or the nonsteroidal antiinflammatory drugs aspirin, metamizol (dipyrone), and indomethacin also increased GPDH mRNA levels in rat spinal cord. The opioid receptor antagonist naloxone completely blocked morphine induction of GPDH but had no effect on GPDH induction by noxious heat stimulation or metamizol treatment, implicating different mechanisms of GPDH induction. Nevertheless, in all cases, induction of the GPDH gene requires adrenal steroids and new protein synthesis, as the induction was blocked in adrenalectomized rats and by cycloheximide treatment, respectively. Our results suggest that the induction of the GPDH gene upon peripheral noxious stimulation is related to the endogenous response to pain as it is mimicked by exogenously applied analgesic drugs.

DREAM is a Ca2+-regulated transcriptional repressor.

Fluxes in amounts of intracellular calcium ions are important determinants of gene expression. So far, Ca2+-regulated kinases and phosphatases have been implicated in changing the phosphorylation status of key transcription factors and thereby modulating their function. In addition, direct effectors of Ca2+-induced gene expression have been suggested to exist in the nucleus, although no such effectors have been identified yet. Expression of the human prodynorphin gene, which is involved in memory acquisition and pain, is regulated through its downstream regulatory element (DRE) sequence, which acts as a location-dependent gene silencer. Here we isolate a new transcriptional repressor, DRE-antagonist modulator (DREAM), which specifically binds to the DRE. DREAM contains four Ca2+-binding domains of the EF-hand type. Upon stimulation by Ca2+, DREAM's ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca2+. In addition to the prodynorphin promoter, DREAM represses transcription from the early response gene c-fos. Thus, DREAM represents the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator.

Protein kinase A-dependent derepression of the human prodynorphin gene via differential binding to an intragenic silencer element.

Induction of the prodynorphin gene has been implicated in medium and long-term adaptation during memory acquisition and pain. By 5' deletion mapping and site-directed mutagenesis of the human prodynorphin promoter, we demonstrate that both basal transcription and protein kinase A (PKA)-induced transcription in NB69 and SK-N-MC human neuroblastoma cells are regulated by the GAGTCAAGG sequence centered at position +40 in the 5' untranslated region of the gene (named the DRE, for downstream regulatory element). The DRE repressed basal transcription in an orientation-independent and cell-specific manner when placed downstream from the heterologous thymidine kinase promoter. Southwestern blotting and UV cross-linking experiments with nuclear extracts from human neuroblastoma cells or human brain revealed a protein complex of approximately 110 kDa that specifically bound to the DRE. Forskolin treatment reduced binding to the DRE, and the time course paralleled that for an increase in prodynorphin gene expression. Our results suggest that under basal conditions, expression of the prodynorphin gene is repressed by occupancy of the DRE site. Upon PKA stimulation, binding to the DRE is reduced and transcription increases. We propose a model for human prodynorphin activation through PKA-dependent derepression at the DRE site.

Metamizol potentiates morphine effects on visceral pain and evoked c-Fos immunoreactivity in spinal cord.

In a model of visceral pain consisting of intraperitoneal injection of acetic acid (writhing test), simultaneous administration of subanalgesic doses of metamizol (150 mg/kg) and morphine (0.2 mg/kg) resulted in a potent analgesia (19 +/- 1 vs. 2.3 +/- 0.8 writhes; P < 0.05). While the analgesic effect of morphine (2 mg/kg) was antagonized by naloxone (1 mg/kg), the opioid antagonist did not reverse the analgesia induced by the combination of metamizol and morphine. Potentiation by metamizol was also observed as a bilateral decrease in stimulus-evoked c-Fos induction in superficial laminas (I-II) of the dorsal spinal cord after drug combination compared to single administration (66.5 +/- 2.2 vs. 80.7 +/- 4.2; P < 0.05). Conversely, the number of nuclei immunostained with an antibody that recognizes all proteins of the Fos family was not modified by the same dose combination compared to single treatment (21.1 +/- 1.3 vs. 20.2 +/- 1.2). Furthermore, in a model of somatic pain consisting of peripheral thermal stimulation of the paws, simultaneous administration of metamizol (100-250 mg/kg) and morphine (0.5 mg/kg) failed to modify flexor reflex latency.

Stimulus-specific hierarchy of enhancer elements within the rat prodynorphin promoter.

Induction of the prodynorphin gene occurs in a tissue-specific manner following different physiological stimuli. Using electrophoretic mobility shift assays, we studied the relative activity of the five major regulatory sites in the rat prodynorphin promoter. Prodynorphin cyclic AMP-responsive element 2 (DynCRE2), DynCRE3, and the noncanonical prodynorphin AP-1 (ncDynAP-1) regulatory sites control, in a coordinated manner, prodynorphin induction in the spinal cord after noxious stimulation, whereas prodynorphin up-regulation in supraoptic neurons is regulated predominantly by the ncDynAP-1. Conversely, prodynorphin transactivation in the ovaries upon gonadotropin stimulation is controlled by DynCRE1 and DynCRE3. Our results support the idea that stimulus-specific changes in nuclear proteins establish a functional hierarchy among regulatory sites in the prodynorphin promoter and provide further insight in the molecular mechanisms that govern prodynorphin gene regulation.

Identification and functional characterization of a K+ channel alpha-subunit with regulatory properties specific to brain.

The physiological diversity of K+ channels mainly depends on the expression of several genes encoding different alpha-subunits. We have cloned a new K+ channel alpha-subunit (Kv2.3r) that is unable to form functional channels on its own but that has a major regulatory function. Kv2.3r can coassemble selectively with other alpha-subunits to form functional heteromultimeric K+ channels with kinetic properties that differ from those of the parent channels. Kv2.3r is expressed exclusively in the brain, being concentrated particularly in neocortical neurons. The functional expression of this regulatory alpha-subunit represents a novel mechanism without precedents in voltage-gated channels, which might contribute to further increase the functional diversity of K+ channels necessary to specify the intrinsic electrical properties of individual neurons.

Peripheral noxious stimulation induces CREM expression in dorsal horn: involvement of glutamate.

Peripheral noxious stimulation is known to trigger signalling cascades in neurons of the spinal cord. The response to pain and stress at the level of gene expression involves transcriptional activation of several cyclic AMP responsive genes. Here, we show induction of the CREM (cyclic-AMP responsive element modulator) gene in distinct subpopulations of spinal cord neurons upon thermal noxious stimulation. The addition of forskolin or glutamate to cultured spinal cord neurons results in the induction of the CREM isoform, ICER (Inducible cyclic-AMP Early Repressor), a powerful repressor of cAMP-induced transcription. Overexpression of ICER in cultured spinal cord neurons results in the repression of the c-fos and c-jun promoters induced by forskolin and glutamate. On this basis, we postulate that early activation of ICER in spinal cord participates in the attenuation of early gene induction following noxious stimulation.

Experimental brain glioma: growth arrest and destruction by a blood-group-related tetrasaccharide.

A synthetic tetrasaccharide (TS4), structurally related to blood groups, inhibited the proliferation of the C6 glioma cells in culture and the growth of tumors formed after intracerebral transplantation of C6 cells. TS4-treated tumors were substantially smaller than controls, as expected from TS4 cytostatic action on C6 glioma cells in culture. However, in vivo treatment also caused extensive tumor destruction. This effect appeared to be caused by indirectly, either by activation of natural killer cells, cytotoxic lymphocytes, or by inhibition of tumor vascularization. Enhanced antigenicity of TS4-treated glioma may be related to the increased expression of connexin 43 observed in glioma cell cultures treated with the oligosaccharide. Because concentrations of up to 20 mg/ml of TS4 were not toxic for normal neuronal or glial cells, specific oligosaccharides such as TS4 offer the possibility of selective tumor treatment.

Functional N-methyl-D-aspartate receptors in clonal rat phaeochromocytoma cells.

1. To characterize from a molecular and functional point of view the endogenous NMDA receptors expressed by phaeochromocytoma (PC12) cells, experiments involving polymerase chain reaction (PCR) amplification, Western blotting and patch-clamp analysis of undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells were performed. 2. Analysis of PC12 mRNA demonstrated the presence of NMDAR1 and NMDAR2C transcripts. The NMDAR1 subunits lack the amino terminal insert of twenty-one amino acid residues, where as transcripts with and without deletions I and II at the 3' end of the coding region were detected. Thus, NMDA receptors of the PC12 cells might include NMDAR1A, NMDAR1E, NMDAR1C and NMDAR1D subunits. 3. Differentiation by NGF treatment of PC12 cells did not alter mRNA expression for NMDA receptor subunits significantly but induced an increase in both the NMDAR1 protein and the total amount of functional receptors that correlated well with a parallel increase in membrane area. 4. NMDA receptors in differentiated PC12 cells had a high affinity for both glutamate and glycine. These were estimated kinetically as 0.59 microM and 74 nM, respectively. Responses to glutamate or NMDA were non-desensitizing in the presence of saturating glycine, but slowly desensitized with low concentrations of glycine. Currents were completely blocked by D-aminophosphonovalerate (APV), 7-Cl-kynurenate and phencyclidine, and showed a voltage-dependent magnesium blockade. Spermine did not potentiate but inhibited NMDA receptor-mediated responses in a voltage-independent manner. 5. With 0.5 mM Ca2+, single-channel analysis revealed very brief openings (mean open time (t(o)) = 0.42 ms), with at least two conductive states, 55 and 33 pS, both having markedly low open probability. At 2 mM Ca2+, conductances were reduced to 39 and 19 pS, without an effect in open probability or mean open time. 6. The functional properties of NMDA receptors in PC12 cells were very similar to those described for NMDAR1A-NMDAR2C heteromers recombinantly expressed. The PC12 cell line provides a simple and reproducible system to analyse some specific NMDA receptor properties.

Expression of the bovine striatal D2 receptor, but not the D1 receptor, in bovine adrenal medulla.

At 37 degrees, the specific binding of [3H]SCH23390 to purified adrenal medullary plasma membranes accounted for only 20% of total binding. At 4 degrees, the binding did not saturate; therefore, equilibrium binding constants could not be estimated. Similar results were obtained with 125I-SCH23982, a ligand that exhibits 25-fold higher specific activity, compared with [3H]SCH23390. Of 11 dopamine receptor ligands used, only (+)-SCH23390 and (+/-)-SKF83566 inhibited the binding of [3H]SCH23390, but with very low affinities (IC50 values of 446 and 635 nM, respectively). In striatal membranes, binding of [3H]SCH23390 and of 125I-SCH23982 followed saturation isotherms. [3H]SCH23390 exhibited a Kd of 383 pM and a Bmax of 479 fmol/mg of protein, and 125I-SCH23982 exhibited a Kd of 664 pM and a Bmax of 453 fmol/mg of protein. The radioligand was displaced by the D1-selective compounds (+)-SCH23390 (IC50 of 3 nM), (+/-)-SKF83556 (IC50 of 5 nM), and (+)-SKF38393 (IC50 of 17 nM); spiperone and quinpirole were ineffective. [3H]Spiperone binding to bovine striatal and adrenal medullary plasma membranes exhibited similar characteristics, compatible with a typical D2 receptor. Northern blot analysis revealed the presence of D1 receptor mRNA in poly(A)+ RNA preparations from bovine brain striatum. When Northern blots containing poly(A)+ from bovine adrenal medulla were probed, no specific hybridization band for D1 receptors was observed; in contrast, a band of the expected size for D2 receptors was obtained. Similar results were obtained with in situ hybridization techniques and with more sensitive reverse transcription-polymerase chain reaction methods. The data support the idea that the peripheral D2 receptor present in bovine adrenal medulla is similar to striatal D2 receptors; in contrast, striatal D1 receptors do not seem to have a counterpart in bovine adrenal medullary tissues.

Differential effect of thyroid hormone on NGFI-A gene expression in developing rat brain.

NGFI-A is an immediate early gene that is rapidly activated in quiescent cells by mitogens or in postmitotic neurons after depolarization. We have previously shown that the expression of NGFI-A in the developing rat brain is under the control of thyroid hormone. Now we report, by means of in situ hybridization histochemistry, the differential effect of thyroid hormone on NGFI-A expression in distinct brain regions depending on the developmental stage. NGFI-A messenger RNA (mRNA) content was analyzed in the piriform cortex, striatum, hippocampus, and cerebral cortex of control, hypothyroid, and T3-injected hypothyroid rats at birth and on postnatal days 5 and 15. In the newborn rats, experimental hypothyroidism is associated with reduced levels of NGFI-A mRNA in most of the brain regions studied. On postnatal day 15, the difference in NGFI-A expression between control and hypothyroid rats is less apparent in the striatum or no longer present in the piriform cortex and the hippocampus. In the cerebral cortex, hypothyroidism is associated with reduced levels of NGFI-A mRNA on postnatal day 15. The dentate gyrus is always insensitive to the thyroidal state. Administration of T3 accelerates the recovery of NGFI-A mRNA in 5- and 15-day-old rats. However, in newborn rats, the effect of the hormone is noticeable only in the piriform cortex. We also show that the reduced level of NGFI-A mRNA in hypothyroidism is accompanied by a reduction in the protein level. Convulsions induced by pentylenetetrazole administration resulted in an increased expression of the NGFI-A gene, which is of similar magnitude in control and hypothyroid rats.

VIP gene expression in rat thymus and spleen.

Vasoactive intestinal peptide (VIP) is a neuropeptide with immunomodulatory properties. In the present study, we demonstrate VIP gene expression in cells of both thymus and spleen in the rat by in situ hybridization. In thymus sections, hybridization signal for VIP mRNA was found in cells in corticomedullary and medulla regions. In the spleen, cells were labeled at the outer area on the periarteriolar lymphoid sheath of the white pulp. Hybridization signal appeared to be in lymphoid cells. These findings suggest that lymphoid cells might produce VIP, which, if released, could exert a paracrine action on central and peripheral lymphoid organs. We suggest that VIP participates in the bidirectional communication between the nervous and the immune systems.

Functional kainate-selective glutamate receptors in cultured hippocampal neurons.

Glutamate mediates fast synaptic transmission at the majority of excitatory synapses throughout the central nervous system by interacting with different types of receptor channels. Cloning of glutamate receptors has provided evidence for the existence of several structurally related subunit families, each composed of several members. It has been proposed that KA1 and KA2 and GluR-5, GluR-6, and GluR-7 families represent subunit classes of high-affinity kainate receptors and that in vivo different kainate receptor subtypes might be constructed from these subunits in heteromeric assembly. However, despite some indications from autoradiographic studies and binding data in brain membranes, no functional pure kainate receptors have so far been detected in brain cells. We have found that early after culturing, a high percentage of rat hippocampal neurons express functional, kainate-selective glutamate receptors. These kainate receptors show pronounced desensitization with fast onset and very slow recovery and are also activated by quisqualate and domoate, but not by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate. Our results provide evidence for the existence of functional glutamate receptors of the kainate type in nerve cells, which are likely to be native homomeric GluR-6 receptors.

Induction of CREM activator proteins in spermatids: down-stream targets and implications for haploid germ cell differentiation.

The cAMP-responsive element modulator (CREM) gene encodes a family of transcriptional regulators that bind to promoter sequences activated by increased intracellular cAMP levels. Both activators and repressors are generated by alternative splicing and alternative translational initiation. During the development of male germ cells, there is a switch in the transcripts generated by CREM. Specifically, from the prophase of meiosis, there is an increase in the CREM tau activator transcript. Here we present results showing that expression of the CREM activator protein is restricted to postmeiotic germ cells. We show that CREM tau is efficiently phosphorylated at a serine residue at position 117 by the protein kinase-A endogenous to germ cells, indicating that it constitutes a natural target of the adenylyl cyclase pathway during spermatogenesis. Phosphorylation of serine-117 turns CREM tau into a powerful activator. The rise in CREM tau protein coincides with the transcriptional activation of several genes. We show that CREM tau efficiently binds to CREs present in the promoters of these genes, suggesting that they could constitute down-stream targets of CREM. We have analyzed in more detail the regulation of one of these genes, the male germ cell-specific RT7. The RT7 promoter is cAMP inducible and activated by CREM tau in transfection assays. The RT7 promoter is efficiently transcribed in vitro with nuclear extracts from seminiferous tubules. CREM-specific antibodies block RT7 in vitro transcription, implicating a role for CREM tau in a cascade of transcriptional events during spermatogenesis.

Nuclear Fos domains in transcriptionally activated supraoptic nucleus neurons.

This study has analysed by light and electron microscopy immunolocalization the nuclear pattern of distribution of Fos-related proteins in supraotic neurons. Two experimental models of transcriptional activation have been used: sustained, global transcriptional activation, at relatively near physiological conditions, by six days of chronic intermittent salt loading; and superinduction of c-fos gene by this salt loading regime plus cycloheximide treatment for 4 h. In the first condition, the ultrastructural analysis showed a distribution of Fos-like immunoreactivity on the reticular network of dispersed chromatin that extends between the nucleolar surface and the nuclear envelope, whereas the Fos-negative adjacent interchromatin spaces appeared rich in interchromatin granules by using a cytochemical staining for ribonucleoproteins. The nucleolus associated heterochromatin, fibrillar centers of the nucleolus and coiled bodies were free of immunoreactivity. This immunoelectron pattern seems to indicate that active genes containing activator protein-1 and cyclic AMP response element recognition sites are extensively distributed in euchromatin regions and suggests that the Fos-positive nuclear domains correspond to the actively transcribing chromatin regions, at least in supraoptic neurons. It also suggests that these Fos-positive transcription domains are complementary to adjacent ribonucleoprotein-rich interchromatin spaces which are involved in the processing and splicing of pre-messenger RNA. Moreover, the absence of immunoreactivity on the fibrillar centers, the sites of pre-ribosomal RNA synthesis, suggests that the Fos protein complexes are not involved in regulating the expression of ribosomal RNA genes. Following superinduction of c-fos gene by osmotic stimulation plus cycloheximide treatment, a conspicuous Fos-like immunoreactivity was detected in dispersed chromatin regions, whereas the heterochromatin masses, nucleoli and coiled bodies showed no immunoreaction. Moreover, this treatment induced the formation of nuclear "dense bodies" of a fibrillar nature which were free of immunolabelling. Since Fos proteins are known to be short-lived, the expression of these nuclear constituents, under conditions of protein synthesis inhibition induced by the cycloheximide, suggests the stabilization of chromatin-bound Fos complexes or, alternatively, a preferential synthesis of Fos proteins.