Transcription Factor Cyclic AMP Response Research Articles

Role of basic region leucine zipper transcription factors cyclic AMP response element binding protein (CREB), CREB2, activating transcription factor 2 and CAAT/enhancer binding protein α in cyclic AMP response element‐mediated transcription

The transcription factor cAMP response element binding protein (CREB), a member of the basic region leucine zipper (bZIP) family of proteins, is the major cAMP response element (CRE) binding. Other bZIP proteins, including CREB2, activating transcription factor 2 (ATF2), or CAAT/enhancer binding protein (C/EBP) have been reported to transactivate CRE-containing genes or to interfere with transactivation by CREB. We have designed a simple transactivation assay using expression of either a constitutively active CREB mutant or a nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase. In both cases, a striking stimulation of transcription of CRE-containing reporter genes was observed in noradrenergic locus coeruleus-like CATH.a cells. In addition, a constitutively active mutant of ATF2 specifically transactivated a secretogranin II promoter/luciferase reporter gene, but had no effect on the tyrosine hydroxylase promoter. In contrast, CREB2 and C/EBPalpha did not transactivate CRE-containing reporter genes, indicating that these bZIP proteins target distinct genetic elements. Experiments involving dominant-negative bZIP mutants revealed that CREB does not heterodimerize with CREB2, ATF2, c-Jun or C/EBP. Rather, CREB and ATF2 compete for binding to the CRE, and are independently able to up-regulate transcription of genes containing CRE motifs in their regulatory regions.

L-type Ca 2+ channel blockers promote Ca 2+ accumulation when dopamine receptors are activated in striatal neurons

Dopamine (DA) receptor-mediated signal transduction and gene expression play a central role in many brain disorders from schizophrenia to Parkinson's disease to addiction. While trying to evaluate the role of L-type Ca 2+ channels in dopamine D 1 receptor-mediated phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB), we found that activation of dopamine D 1 receptors alters the properties of L-type Ca 2+ channel inhibitors and turns them into facilitators of Ca 2+ influx. In D 1 receptor-stimulated neurons, L-type Ca 2+ channel blockers promote cytosolic Ca 2+ accumulation. This leads to the activation of a molecular signal transduction pathway and CREB phosphorylation. In the absence of dopamine receptor stimulation, L-type Ca 2+ channel blockers inhibit CREB phosphorylation. The effect of dopamine on L-type Ca 2+ channel blockers is dependent on protein kinase A (PKA), suggesting that protein phosphorylation plays a role in this phenomenon. Because of the adverse effect of activated dopamine receptors on L-type Ca 2+ channel blocker action, the role of L-type Ca 2+ channels in the dopamine D 1 receptor signal transduction pathway cannot be assessed with pharmacological tools. However, with antisense technology, we demonstrate that L-type Ca 2+ channels contribute to D 1 receptor-mediated CREB phosphorylation. We conclude that the D 1 receptor signal transduction pathway depends on L-type Ca 2+ channels to mediate CREB phosphorylation.

Phosphatidylinositol 3‐kinase and Akt effectors mediate insulin‐like growth factor‐I neuroprotection in dorsal root ganglia neurons

Insulin-like growth factor-I (IGF-I) protects neurons of the peripheral nervous system from apoptosis, but the underlying signaling pathways are not well understood. We studied IGF-I mediated signaling in embryonic dorsal root ganglia (DRG) neurons. DRG neurons express IGF-I receptors (IGF-IR), and IGF-I activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. High glucose exposure induces apoptosis, which is inhibited by IGF-I through the PI3K/Akt pathway. IGF-I stimulation of the PI3K/Akt pathway phosphorylates three known Akt effectors: the survival transcription factor cyclic AMP response element binding protein (CREB) and the pro-apoptotic effector proteins glycogen synthase kinase-3beta (GSK-3beta) and forkhead (FKHR). IGF-I regulates survival at the nuclear level through accumulation of phospho-Akt in DRG neuronal nuclei, increased CREB-mediated transcription, and nuclear exclusion of FKHR. High glucose increases expression of the pro-apoptotic Bcl protein Bim (a transcriptional target of FKHR). However, IGF-I does not regulate Bim or anti-apoptotic Bcl-xL protein expression levels, which suggests that IGF-I neuroprotection is not through regulation of their expression. High glucose also induces loss of the initiator caspase-9 and increases caspase-3 cleavage, effects blocked by IGF-I. These data suggest that IGF-I prevents apoptosis in DRG neurons by regulating PI3K/Akt pathway effectors, including GSK-3beta, CREB, and FKHR, and by blocking caspase activation.

The protein phosphatase 1/2A inhibitor okadaic acid increases CREB and Elk-1 phosphorylation and c-fos expression in the rat striatum in vivo.

Activation of group I metabotropic glutamate receptors (mGluRs) up-regulates transcription factor cyclic AMP response element-binding protein (CREB) and Elk-1 phosphorylation via extracellular signal-regulated kinase 1/2 (ERK1/2) in the striatum in vivo. Protein phosphatase 1/2A further regulates immediate early gene expression by inactivating (dephosphorylating) CREB. In this study, using semi-quantitative immunohistochemical and western blot analyses and in situ hybridization histochemistry, we found that intrastriatal infusion of the protein phosphatase 1/2A inhibitor okadaic acid (0.005, 0.05 and 0.5 nmol) increased CREB and Elk-1 phosphorylation and c-Fos immunoreactivity in the injected dorsal striatum in a dose-dependent manner. In addition, okadaic acid (0.05 and 0.5 nM) increased c-fos mRNA expression in the dorsal striatum in a dose-dependent manner. Intrastriatal infusion of the group I agonist 3,5-dihydroxyphenylglycine (DHPG) at 100 and 250 nM also increased CREB and Elk-1 phosphorylation. Pre-treatment of okadaic acid (0.05 nm) did not alter DHPG-induced increases in the phosphorylation of the two transcription factors. These data suggest that protein phosphatase 1/2A in striatal neurons is tonically active in dephosphorylating CREB and Elk-1 and thus suppressing constitutive c-fos mRNA and protein expression. Inhibition of the phosphatase 1/2A may contribute to the group I mGluR-regulated phosphorylation of these transcription factors and c-fos expression.

Exercise reverses the harmful effects of consumption of a high-fat diet on synaptic and behavioral plasticity associated to the action of brain-derived neurotrophic factor

A diet high in total fat (HF) reduces hippocampal levels of brain-derived neurotrophic factor (BDNF), a crucial modulator of synaptic plasticity, and a predictor of learning efficacy. We have evaluated the capacity of voluntary exercise to interact with the effects of diet at the molecular level. Animal groups were exposed to the HF diet for 2 months with and without access to voluntary wheel running. Exercise reversed the decrease in BDNF and its downstream effectors on plasticity such as synapsin I, a molecule with a key role in the modulation of neurotransmitter release by BDNF, and the transcription factor cyclic AMP response element binding protein (CREB), important for learning and memory. Furthermore, we found that exercise influenced the activational state of synapsin as well as of CREB, by increasing the phosphorylation of these molecules. In addition, exercise prevented the deficit in spatial learning induced by the diet, tested in the Morris water maze. Furthermore, levels of reactive oxygen species increased by the effects of the diet were decreased by exercise. Results indicate that exercise interacts with the same molecular systems disrupted by the HF diet, reversing their effects on neural function. Reactive oxygen species, and BDNF in conjunction with its downstream effectors on synaptic and neuronal plasticity, are common molecular targets for the action of the diet and exercise. Results unveil a possible molecular mechanism by which lifestyle factors can interact at a molecular level, and provide information for potential therapeutic applications to decrease the risk imposed by certain lifestyles.

Opposite regulation by typical and atypical anti-psychotics of ERK1/2, CREB and Elk-1 phosphorylation in mouse dorsal striatum.

The two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), are involved in the control of gene expression via phosphorylation and activation of the transcription factors cyclic AMP response element binding protein (CREB) and Elk-1. Here, we have examined the effect of haloperidol and clozapine, two anti-psychotic drugs, and eticlopride, a selective dopamine D2 receptor antagonist, on the state of phosphorylation of ERK1/2, CREB and Elk-1, in the mouse dorsal striatum. Administration of the typical anti-psychotic haloperidol stimulated the phosphorylation of ERK1/2, CREB and Elk-1. Virtually identical results were obtained using eticlopride. In contrast, the atypical anti-psychotic clozapine reduced ERK1/2, CREB and Elk-1 phosphorylation. This opposite regulation was specifically exerted by haloperidol and clozapine on ERK, CREB, and Elk-1 phosphorylation, as both anti-psychotic drugs increased the phosphorylation of the dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32) at the cyclic AMP-dependent protein kinase (PKA) site. The activation of CREB and Elk-1 induced by haloperidol appeared to be achieved via different signalling pathways, as inhibition of ERK1/2 activation abolished the stimulation of Elk-1 phosphorylation without affecting CREB phosphorylation. This study shows that haloperidol and clozapine induce distinct patterns of phosphorylation in the dorsal striatum. The results provide a novel biochemical paradigm elucidating the molecular mechanisms underlying the distinct therapeutic actions of typical and atypical anti-psychotic agents.

Oxidative stress‐mediated down‐regulation of bcl‐2 promoter in hippocampal neurons

Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.

Activation of the cyclic amp response element-binding protein signaling pathway in the olfactory bulb is required for the acquisition of olfactory aversive learning in young rats

Long-term memory formation requires both gene expression and protein synthesis. Phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB) is thought to be important in processes underlying long-term memory. To clarify the role of CREB in olfactory aversive learning in young rats, we carried out behavioral pharmacology and Western blot analyses. On postnatal day 11, oligodeoxynucleotides were infused directly into the bilateral olfactory bulbs through cannulae implanted prior to training in a classical conditioning paradigm with citral odor and foot shock. On the following day the odor preference test was performed. After training, saline-infused animals spent significantly shorter time over the citral odor zone. Infusion of CREB antisense oligodeoxynucleotides 6 h before or during training, however, prevented olfactory aversive learning without affecting memory retention 1 h after training. CREB scrambled oligodeoxynucleotides infusions had no effect on olfactory learning. When infused 6 h after training, none of oligodeoxynucleotides had an effect on time spent over the odor zone. Using Western blotting, we analyzed CREB in nuclear extracts obtained from the young rats after training. Marked increases in phosphorylated CREB were sustained from 10 to 360 min after the odor-shock pairing in animals which were subjected to both, in comparison with levels 30 min in animals which were subjected to odor only or no stimulation. Total CREB levels showed no differences among groups. Infusion of CREB antisense oligodeoxynucleotides significantly reduced the expression of phosphorylated and total CREBs in the olfactory bulb. These results show that the synthesis and phosphorylation of CREB are required for the acquisition of olfactory aversive learning in young rats, and that this requirement for the CREB signaling pathway has a critical time window.

Excitatory actions of GABA increase BDNF expression via a MAPK-CREB-dependent mechanism--a positive feedback circuit in developing neurons.

During early neuronal development, GABA functions as an excitatory neurotransmitter, triggering membrane depolarization, action potentials, and the opening of plasma membrane Ca(2+) channels. These excitatory actions of GABA lead to a number of changes in neuronal structure and function. Although the effects of GABA on membrane biophysics during early development have been well documented, little work has been done to examine the possible mechanisms underlying GABA-regulated plastic changes in the developing brain. This study focuses on GABA-regulated kinase activity and transcriptional control. We utilized a combination of Western blotting and immunocytochemical techniques to examine two potential downstream pathways regulated by GABA excitation: the p42/44 mitogen-activated protein kinase (MAPK) cascade and the transcription factor cyclic AMP response element binding protein (CREB). During early development of cultured hypothalamic neurons (5 days in vitro), stimulation with GABA triggered activation of the MAPK cascade and phosphorylation of CREB at Ser 133. These effects were mediated by the GABA(A) receptor, since administration of the GABA(A) receptor-specific agonist muscimol (50 microM) triggered pathway activation, and pretreatment with the GABA(A)-receptor specific antagonist bicuculline (20 microM) blocked pathway activation. Immunocytochemistry revealed a spatial and temporal correlation between activation of the MAPK cascade and CREB phosphorylation. Pretreatment with the MAPK/ERK kinase (MEK) inhibitor U0126 (10 microM) attenuated CREB phosphorylation, indicating that the MAPK pathway regulates that activation state of CREB. In contrast to the excitatory effects observed during early development, in more mature neurons, GABA functions as an inhibitory transmitter. Consistent with this observation, GABA(A) receptor activation did not stimulate MAPK cascade activation or CREB phosphorylation in mature cultures (18 days in vitro). To determine whether GABA(A) receptor activation during early development stimulates gene expression, we examined the inducible expression of the neurotrophin brain-derived neurotrophic factor (BDNF). Both GABA and muscimol stimulated BDNF expression, and pretreatment with U0126 attenuated GABA-induced BDNF expression. Whole cell electrophysiological recording was used to assess the effects of BDNF on GABA release. BDNF (100 ng/ml) dramatically increased the frequency of excitatory GABAergic spontaneous postsynaptic currents. Together, these data suggest a positive excitatory feedback loop between GABA and BDNF expression during early development, where GABA facilitates BDNF expression, and BDNF facilitates the synaptic release of GABA. Signaling via the MAPK cascade and the transcription factor CREB appear to play a substantial role in this process.

Mammalian melatonin receptors: molecular biology and signal transduction.

The pineal hormone, melatonin, is an important regulator of seasonal reproduction and circadian rhythms. Its effects are mediated via high-affinity melatonin receptors, located on cells of the pituitary pars tuberalis (PT) and suprachiasmatic nucleus (SCN), respectively. Two subtypes of mammalian melatonin receptors have been cloned and characterized, the MT1 (Mel(1a)) and the MT2 (Mel(1b)) melatonin receptor subtypes. Both subtypes are members of the seven-transmembrane G protein-coupled receptor family. By using recombinant melatonin receptors it has been shown that the MT1 melatonin receptor is coupled to different G proteins that mediate adenylyl cyclase inhibition and phospholipase C beta activation. The MT2 receptor is also coupled to inhibition of adenylyl cyclase and additionally it inhibits the soluble guanylyl cyclase pathway. In mice with a targeted deletion of the MT1 receptor, the acute inhibitory effects of melatonin on SCN multiunit activity are completely abolished, while the phase-shifting responses to melatonin (given in physiological concentrations) appear normal. Furthermore, melatonin inhibits the phosphorylation of the transcription factor cyclic AMP response element binding protein, induced by the pituitary adenylate cyclase-activating polypeptide in SCN cells predominantly via the MT1 receptor. However, a functional MT2 receptor in the rodent SCN is partially able to compensate for the absence of the MT1 receptor in MT1 receptor-deficient mice. These findings indicate redundant and non-redundant roles of the receptor subtypes in regulating SCN function. In the PT, a functional MT1 receptor is essential for the rhythmic synthesis of the clock gene product mPER1. Melatonin produces a long-lasting sensitization of adenylyl cyclase and thus amplifies cyclic AMP signaling when melatonin levels decline at dawn. This action of melatonin amplifies gene expression rhythms in the PT and provides a mechanism for reinforcing rhythmicity in peripheral tissues which themselves lack the capacity for self-sustained oscillation. Mice with targeted deletion of melatonin receptor subtypes provide an excellent model to understand cellular mechanisms through which melatonin modulates circadian and photoperiodic rhythmicity.

Ginkgo biloba extract : Cognitive enhancer or antistress buffer

Constituents extracted from the leaves of the Ginkgo biloba tree possess beneficial properties that may buffer the aging nervous system from deterioration due to oxidative stress. In the present investigation, a standardized extract of G. biloba (EGb 761) or an equal volume of the vehicle was administered (100 mg/kg/day) to senescent (20-month) C57BL/6 male mice for up to 82 consecutive days. Animals were tested twice in the Morris water maze (MWM) after 28 and 70 days of treatment. No differences were observed in acquisition or retention of performance on the water maze. Elevated-plus maze (EPM) trials were conducted prior to and subsequent to the chronic treatment regimen. Marked baseline differences in plus-maze performance were present in the first experiment. A second experiment used a matched-pairs design to minimize preexisting differences. Results supported the hypothesis that EGb 761 may serve as an antistress buffer, attenuating the increase in anxiety typically observed in animals after cold water exposure. Tissue samples from the hippocampus and cortex were analyzed by Western blot for the transcription factor cyclic-AMP response element binding (CREB) protein. EGb 761 had no significant effect on immunoreactivity to CREB from either the hippocampus or the cerebral cortex.

Influence of exercise intensity on ERK/MAP kinase signalling in human skeletal muscle.

The mitogen-activated protein (MAP) kinase pathways have been highlighted as a possible link between exercise and adaptive changes in skeletal muscle. In this study, the effect of exercise intensity on the activation of the ERK/MAP kinase pathway was investigated in human skeletal muscle. One-leg exercise at low (40% maximal oxygen consumption, VO2max for 30 min) and high (75% VO2max for 30 min) intensity resulted in 11.5+8. I-fold and 39.7+/-6.3-fold (mean +/-SEM) increases in ERK1/2 phosphorylation (P<0.001), respectively. The phosphorylation of MEK1/2, the upstream kinase of ERK1/2, increased with exercise intensity (P<0.05) to 2.5+/-0.9 and 4.8+/-1.1 times the basal level at the low and high intensity, respectively. The statistical analysis revealed a systematic difference between basal, low and high intensity exercise levels for both kinases. There was no change in the phosphorylation of either kinase in the non-exercised leg. The phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB), a possible downstream target of the ERK/MAP kinase signalling pathway, was unaffected by exercise. The phosphorylation of ERK1/2 was significantly higher in purified freeze-dried compared to crude wet muscle after exercise, whereas the opposite pattern was observed for CREB. In conclusion, phosphorylation of ERK1/2 and MEK1/2 increases in an exercise intensity-dependent manner in human skeletal muscle and this seems to originate in the muscle fibres themselves.

Role of mitogen-activated protein kinase cascades in mediating lipopolysaccharide-stimulated induction of cyclooxygenase-2 and IL-1 beta in RAW264 macrophages.

LPS stimulation of RAW264 macrophages triggered the activation of mitogen- and stress-activated protein kinases-1 and -2 (MSK1, MSK2) and their putative substrates, the transcription factors cyclic AMP response element-binding protein (CREB) and activating transcription factor-1 (ATF1). The activation of MSK1/MSK2 was prevented by preincubating the cells with a combination of two drugs that suppress activation of the classical mitogen-activated protein kinase cascade and stress-activated protein kinase/p38, respectively, but inhibition was only partial in the presence of either inhibitor. The LPS-stimulated activation of CREB and ATF1, the transcription of the cyclooxygenase-2 (COX-2) and IL-1 beta genes (the promoters of which contain a cyclic AMP response element), and the induction of the COX-2 protein were prevented by the same drug combination, as well as by Ro 318220 or H89, potent inhibitors of MSK1/MSK2. Two other transcription factors, C/EBP beta and NF-kappa B, have been implicated in the transcription of the COX-2 gene. However, PD 98059 and/or SB 203580 did not prevent the LPS-induced increase in the level of the transcription factor C/EBP beta, and none of the four inhibitors used in this study prevented the activation of NF-kappa B. Our results demonstrate that two different mitogen-activated protein kinase cascades are rate limiting for the LPS-induced activation of CREB/ATF1 and the transcription of the COX-2 and IL-1 beta genes. They also suggest that MSK1 and MSK2 may play a role in these processes and hence are potential targets for the development of novel antiinflammatory drugs.

Use of a drug-resistant mutant of stress-activated protein kinase 2a/p38 to validate the in vivo specificity of SB 203580

Stress-activated protein kinase 2a, also called p38, is inhibited by SB 203580 and this drug has been used widely to implicate this enzyme in the regulation of many physiological processes. Here, we introduce a novel method of general application, which can be used to establish whether the effects of SB 203580 are mediated via inhibition of stress-activated protein kinase 2a/p38 or whether they result from ‘non-specific’ effects. Four events thought to occur upon activation of stress-activated protein kinase 2a/p38 have been established unequivocally. These are the activation of mitogen-activated protein kinase-activated protein kinase-2 and mitogen- and stress-activated protein kinase-1 and the phosphorylation of their presumed substrates, heat shock protein 27 and the transcription factor cyclic AMP response element binding protein, respectively. In contrast, the SB 203580-induced activation of c-Raf is independent of stress-activated protein kinase 2a/p38 inhibition.

Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective.

The mammalian brain has a high degree of plasticity, with dentate granule cell neurogenesis and glial proliferation stimulated by an enriched environment combining both complex inanimate and social stimulation. Moreover, rodents exposed to an enriched environment both before and after a cerebral insult show improved cognitive performance. One of the most robust associations of environmental enrichment is improved learning and memory in the Morris water maze, a spatial task that mainly involves the hippocampus. Furthermore, clinical evidence showing an association between higher educational attainment and reduced risk of Alzheimer and Parkinson-related dementia indicates that a stimulating environment has positive effects on cerebral health that may provide some resilience to cerebral insults. Here we show that in addition to its effects on neurogenesis, an enriched environment reduces spontaneous apoptotic cell death in the rat hippocampus by 45%. Moreover, these environmental conditions protect against kainate-induced seizures and excitotoxic injury. The enriched environment induces expression of glial-derived neurotrophic factor and brain-derived neurotrophic factor and increases phosphorylation of the transcription factor cyclic-AMP response element binding protein, indicating that the influence of the environment on spontaneous apoptosis and cerebral resistance to insults may be mediated through transcription factor activation and induction of growth factor expression.

Impaired experience-dependent plasticity in barrel cortex of mice lacking the alpha and delta isoforms of CREB.

The transcription factor cyclic-AMP response element binding protein (CREB) has been implicated in long-term plasticity processes in vertebrate and invertebrate species. In the absence of the alpha/delta CREB isoforms, performance is impaired in long-term memory tasks and the long-term maintenance of long-term potentiation (LTP) is impaired in the hippocampus. However, it is not known whether CREB plays a role in neocortical plasticity. Antibodies to CREB revealed that CREB-immunoreactive nuclei are present in all cortical layers but are more numerous in layers II/III, where they composed at least two-thirds the total population of cells. CREB-immunopositive cells were therefore present and densest in the very cortical layers that exhibit experience-dependent plasticity at this age. In order to assess the role of CREB in neocortical plasticity, we studied the effect of vibrissae deprivation on receptive field plasticity in the barrel cortex of mutant mice lacking the alpha/delta isoforms of CREB. A single vibrissa was spared and the others removed for 18 days. In wild-types this caused potentiation of the spared vibrissa response. However, in adult mutants (>6 months) spared vibrissa responses from homozygotes were potentiated less than in any adolescent animals or in adult wild-type littermates. Surround receptive field responses were abnormally large in homozygotes and failed to increase by the same amount as they did in wild-types. In contrast, the alpha/delta CREB mutation had no discernible effect on plasticity in cortical layers II/III of the younger adolescent age group (1-2 months), suggesting that different plasticity processes may operate at this age. Further tests showed that the beta isoform of CREB was up-regulated in the barrel cortex of the alpha/delta CREB knock-outs, suggesting that this subunit may have compensated partly for the loss of the alpha/delta isoforms. These studies suggests that CREB plays a role in experience-dependent plasticity in the adult neocortex.

The Neuron-Glia Signal β Neuregulin Induces Sustained CREB Phosphorylation on Ser-133 in Cultured Rat Schwann Cells

β neuregulins (also called NDF, GGF, ARIA, and heregulins) are neuron-derived molecules that are likely to be responsible for Schwann cell precursor survival, proliferation, and maturationin vivoandin vitro.Although the receptors to which β neuregulins bind have been defined, little is known about the transcription factors these important ligands activate. Using antibodies, quantitative imaging methods and Western blotting, we show that β neuregulin induces a high level of phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB) on Ser-133 in cultured rat Schwann cells and that the phosphorylation is prolonged over several hours. In contrast, neurotrophins, CNTF, FGF-2, EGF, and TGFβ induce little or no phosphorylation of CREB despite the fact that receptors for these factors are present on Schwann cells. As expected CREB phosphorylation was detected following cAMP elevation, and it was also induced by elevation of cytoplasmic Ca2+, endothelin 1, and PDGF-BB. The signal was lower than that seen in response to β neuregulin, and transient, unlike the sustained CREB activation induced by β neuregulin. Our results suggest that the sustained phosphorylation of CREB on Ser-133 may contribute to the broad spectrum of effects that β neuregulins have on cells of the Schwann cell lineage and that the CREB pathway may be important for transduction of neuregulin signals in Schwann cells.

Promoter Activation via a Cyclic AMP Response Element in Vitro

Transcription activation via activating transcription factor cyclic AMP response element binding (ATF/CREB) sites in vitro was explored using transcription and permanganate assay for open complex formation. These sites were used to drive transcription from an adenovirus major late core sequence. Under conditions where activation is strong, 20-50-fold, ATF/CREB is required for preinitiation complexes to reach the open complex stage. Complete opening requires activator, ATP, and initiating nucleotides. In exploration of postinitiation steps, no stimulation of promoter clearance was observed but a modest stimulation of the rate of continuous transcription occurred. High amounts of DNA template, commonly used in in vitro studies, allows some templates to open without activator, but leaves the nucleotide requirements intact. This leads to a drastic lowering of the dependence on ATF/CREB. Taken together, the data indicate that ATF/CREB activates this system primarily by stimulating the formation of functional preinitiation complexes.

CREB: A Major Mediator of Neuronal Neurotrophin Responses

Neurotrophins regulate neuronal survival, differentiation, and synaptic function. To understand how neurotrophins elicit such diverse responses, we elucidated signaling pathways by which brain-derived neurotrophic factor (BDNF) activates gene expression in cultured neurons and hippocampal slices. We found, unexpectedly, that the transcription factor cyclic AMP response element-binding protein (CREB) is an important regulator of BDNF-induced gene expression. Exposure of neurons to BDNF stimulates CREB phosphorylation and activation via at least two signaling pathways: by a calcium/calmodulin-dependent kinase IV (CaMKIV)-regulated pathway that is activated by the release of intracellular calcium and by a Ras-dependent pathway. These findings reveal a previously unrecognized, CaMK-dependent mechanism by which neurotrophins activate CREB and suggest that CREB plays a central role in mediating neurotrophin responses in neurons.

Novel Repeat Elements Direct Rat Proenkephalin Transcription during Spermatogenesis

The developmental program controlling sperm formation occurs in multiple stages that sequentially involve mitosis, meiosis, and spermiogenesis. The transcriptional mechanisms regulating these distinct phases are poorly understood. In particular, while a required role for the germ cell transcription factor cyclic AMP response element modulator-tau during spermiogenesis has recently been demonstrated, the transcriptional mechanisms leading to early haploid cell formation are unknown. The rat and mouse proenkephalin genes are selectively expressed from an alternate, germ cell-specific promoter in meiotic and early haploid cells. In this study, the minimal rat proenkephalin germ line promoter was localized to a 116-bp region encompassing the transcriptional start site region. Further, a proximal 51-bp sequence located in the 5'-flanking region is absolutely required for germ line promoter activity. This 51 bp sequence corresponds to a previously characterized binding element (GCP1) that forms cell-specific complexes with rat spermatogenic cell nuclear factors distinct from cyclic AMP response element binding proteins. Further, GCP1 contains novel direct repeat sequences required for factor binding and transgene expression in spermatogenic cells. These repeat elements are highly similar to sequences within the active regions of other male germ line promoters expressed during meiosis. GCP1 may therefore contain transcriptional elements that participate more generally during meiosis in the differentiation of spermatocytes and early haploid spermatids.