Regulators of A20 (TNFAIP3): new drug-able targets in inflammation.

Persistent activation of the transcription factor Nuclear factor-κB (NF-κB) is central to the pathogenesis of many inflammatory disorders, including those of the lung such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease (COPD). Despite recent advances in treatment, management of the inflammatory component of these diseases still remains suboptimal. A20 is an endogenous negative regulator of NF-κB signaling, which has been widely described in several autoimmune and inflammatory disorders and more recently in terms of chronic lung disorders. However, the underlying mechanism for the apparent lack of A20 in CF, COPD, and asthma has not been investigated. Transcriptional regulation of A20 is complex and requires coordination of different transcription factors. In this review we examine the existing body of research evidence on the regulation of A20, concentrating on pulmonary inflammation. Special focus is given to the repressor downstream regulatory element antagonist modulator (DREAM) and its nuclear and cytosolic action to regulate inflammation. We provide evidence that would suggest the A20-DREAM axis to be an important player in (airway) inflammatory responses and point to DREAM as a potential future therapeutic target for the modification of phenotypic changes in airway inflammatory disorders. A schematic summary describing the role of DREAM in inflammation with a focus on chronic lung diseases as well as the possible consequences of altered DREAM expression on immune responses is provided.

A neuroprotective phase precedes striatal degeneration upon nucleolar stress.

The nucleolus is implicated in sensing and responding to cellular stress by stabilizing p53. The pro-apoptotic effect of p53 is associated with several neurodegenerative disorders, including Huntington's disease (HD), which is characterized by the progressive loss of medium spiny neurons (MSNs) in the striatum. Here we show that disruption of nucleolar integrity and function causes nucleolar stress and is an early event in MSNs of R6/2 mice, a transgenic model of HD. Targeted perturbation of nucleolar function in MSNs by conditional knockout of the RNA polymerase I-specific transcription initiation factor IA (TIF-IA) leads to late progressive striatal degeneration, HD-like motor abnormalities and molecular signatures. Significantly, p53 prolongs neuronal survival in TIF-IA-deficient MSNs by transient upregulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor that inhibits mammalian target of rapamycin signaling and induces autophagy. The results emphasize the initial role of nucleolar stress in neurodegeneration and uncover a p53/PTEN-dependent neuroprotective response.

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.

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+).

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.

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.

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.

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.

Differences in expression of transcription factor AP-1 in human promyelocytic HL-60 cells during differentiation towards macrophages versus granulocytes.

Commitment of HL-60 cells to macrophage or granulocytic differentiation was achieved by incubation with 4 beta-phorbol 12-myristate 13-acetate (PMA) for 30-60 min or with dimethyl sulphoxide (DMSO) for 24 h respectively. The commitment stage towards PMA-induced macrophage differentiation was associated with increases in jun B and c-fos mRNA levels, as well as with an increase in the binding activity of transcription factor AP-1. Nevertheless, gel retardation analysis indicated that the AP-1 activity detected in untreated cells was drastically reduced during the commitment stage of DMSO-induced HL-60 differentiation towards granulocytes. When HL-60 cells were treated with sodium butyrate, which induced monocytic differentiation, a remarkable increase in AP-1 binding activity was detected. Treatment of HL-60 cells with 1 alpha,25-dihydroxyvitamin D3, another monocytic differentiation agent, induced a weak, but appreciable, increase in AP-1 activity. Furthermore, addition of sodium butyrate or 1 alpha,25-dihydroxyvitamin D3 to HL-60 cells induced the expression of c-fos, c-jun, jun B and jun D proto-oncogenes. In contrast, when HL-60 cells were treated with retinoic acid, a granulocytic differentiation inducer, no enhanced AP-1 binding activity was observed, and only a weak increase in jun D mRNA level was detected. These data indicate that formation of AP-1 is not required for the induction of HL-60 differentiation towards granulocytes, whereas induction of monocytic differentiation is correlated with an increase in AP-1 activity. The differential expression of AP-1 activity may be critical in the differentiation of HL-60 cells towards monocytic or granulocytic lineages.

Transcriptional response to cAMP in brain: specific distribution and induction of CREM antagonists.

Changes in cAMP levels are often associated with the modulation of neuronal function. The CREM gene encodes both antagonists and activators of the cAMP-dependent transcriptional response by alternative splicing. CREM transcripts in rat brain show a characteristic pattern of expression, being specific for the inner layer of the cerebral cortex, anterior thalamus, hippocampus, and hypothalamus. Strikingly, the CREM transcripts correspond to the antagonist isoforms in these areas, suggesting a down-regulatory role for CREM in brain; in contrast, the expression of CREM tau and CREB activators is more diffuse and generalized. In the supraoptic nucleus, CREM expression is induced after osmotic stimulus. Importantly, this demonstrates physiological inducibility of CREM, which is novel within the CRE/ATF family.

Molecular mechanisms of pain: serotonin1A receptor agonists trigger transactivation by c-fos of the prodynorphin gene in spinal cord neurons.

By using spinal cord neurons cultured in chemically defined medium, a double labeling procedure, and blockage with antisense oligonucleotides, we show that induction of c-fos and the subsequent transactivation of the prodynorphin gene are coupled events, triggered by serotonin1A receptor agonists. Addition of the specific 1A agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) to the culture, at concentrations similar to that needed for transactivation of the prodynorphin gene, also significantly increases cAMP levels. Furthermore, in rats depleted of serotonin by intrathecal administration of 5,7-dihydroxytryptamine, the induction of prodynorphin after noxious stimulation is dramatically decreased compared with the induction in sham-operated rats. These results suggest that the expression of the prodynorphin gene in spinal cord is under the control of the raphe-spinal efferents containing serotonin.

Narine occlusion decreases basal levels of Fos protein in the cerebral cortex of the lizard Podarcis hispanica.

Immunocytochemical study of cerebral cortex of the lizard Podarcis hispanica using an antibody directed to the M peptide of the rat c-Fos protein showed a distinct pattern of Fos distribution. Abundant Fos-immunoreactive neuronal nuclei were detected in the cell layers of the medial, the dorsal and the lateral cortices, whereas only a few nuclei were found in the cell layer of the dorsomedial cortex. The Fos immunoreactivity was characterized by Western blot analysis of nuclear extracts from lizard brain and showed a distinct band with an apparent molecular weight of 30,000. In band-shift assays, nuclear extracts from lizard brain were shown to contain AP-1 complexes. The basal expression of Fos immunoreactivity is related to sensory olfactory input in the cerebral cortex of the lizard since experiments with olfactory-deprived animals resulted in a complete absence of Fos immunoreactivity in the cortical areas.

Fos-like expression and nuclear size in osmotically stimulated supraoptic nucleus neurons.

This study has analysed by immunocytochemistry the pattern of expression of Fos-related proteins, as well as variations in nuclear size, after the osmotically induced activation of supraoptic nucleus neurons of the rat. In control rats most supraoptic nucleus neurons were Fos-like negative. After acute and chronic dehydration by salt-loading, the number of Fos-like positive neurons increased dramatically. The level of Fos-like immunoreactivity was higher in chronically stimulated rats, and also the neurons of the ventral region of the supraoptic nucleus were more intensely stained than those of the dorsal region. The karyometric analysis was made on electron micrographs. The mean nuclear profile area showed a significant increase in dehydrated rats with respect to the controls (73 +/- 16 microns 2 in those dehydrated for six days vs 54 +/- 13 in controls, mean +/- S.D.). However, no significant differences in this parameter were found when one-day and six-day dehydrated groups were compared. The invagination factor of the nuclear membrane, a nuclear shape indicator, decreased significantly in dehydrated rats, indicating a tendency towards spherical nuclei. It is noteworthy that the nuclear profile perimeter was constant, about 32 microns, in control and osmotically simulated rats. The higher nuclear accumulation of Fos-related antigens after six days of dehydration suggests that in chronically stimulated supraoptic nucleus neurons there is a sustained induction of cell-specific genes. Moreover, the transcription rate of the target genes containing the consensus DNA sequence TGAC/GTCA or c-AMP responsive elements recognition sites may depend upon the nuclear concentration of Fos-related antigens in supraoptic nucleus neurons. Our results also suggest that the initial Fos-related antigen expression and nuclear size increase are triggered concomitantly in supraoptic nucleus neurons after a short period of osmotic stimulation. On the other hand, we propose that nuclear envelope invaginations represent a reservoir of nuclear membrane which allows dynamic changes in nuclear size and shape depending on the metabolic status of the supraoptic nucleus neurons.

Differential expression of the jun family members in rat brain.

The transcription factor AP-1 is phorbol ester-regulated and, as such, is considered to be a nuclear target of the signal transduction pathway involving protein kinase C. AP-1 is constituted by the various products of the jun and fos gene family members. These genes belong to the early response class and are inducible in different ways by growth factors, phorbol esters and depolarization. We studied the transcript distribution of c-jun, junB and junD in the rat brain. Our results show that the transcripts for these three genes are differentially distributed in various neuronal tissues. We also provide evidence for developmentally regulated expression of jun genes in post-natal brain. The spatiotemporal pattern of expression of c-jun, junB and junD offers clues to the understanding of the links between gene regulation and neuronal processes.