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At least 793 records · Page 44Linked to original sources
PubMed2002

Developmental Pb2+ exposure alters NMDAR subtypes and reduces CREB phosphorylation in the rat brain.

In the present study we show that chronic exposure to low levels of lead (Pb(2+)) during development alters the type of N-methyl-D-aspartate receptor (NMDAR) expressed in the developing and young adult rat brain. Ifenprodil inhibition of [3H]MK-801 binding to the NMDAR channel in cortical and hippocampal neuronal membranes expressed high and low affinity components. Previous studies have shown that the high affinity component is associated with NR1/NR2B receptor complexes while the low affinity component is associated with the appearance and insertion of the NR2A subunit to NMDAR complexes. Pb(2+)-exposed rats express a greater number of [3H]MK-801 binding sites associated with the high affinity and low affinity components of ifenprodil inhibition. Further, [3H]ifenprodil saturation isotherms and Scatchard analysis in cortical and hippocampal membranes showed a higher number of binding sites (B(max)) with no change in binding affinity (K(d)) in Pb(2+)-exposed animals relative to controls. Quantitative [3H]MK-801 autoradiography in response to glutamate and glycine provided evidence that NMDAR complexes in Pb(2+)-exposed rat brain were maximally activated in situ. Higher levels of ifenprodil-sensitive binding sites and increased sensitivity to agonists are properties characteristic of NR1/NR2B recombinant receptors. Thus, our results strongly suggest that a greater proportion of the total number of NMDAR are NR1/NR2B receptors in the Pb(2+)-exposed rat brain. This Pb(2+)-induced change in NMDAR subtypes in the rat brain was associated with reduced CREB phosphorylation in cortical and hippocampal nuclear extracts. These findings demonstrate that chronic exposure to environmentally relevant levels of Pb(2+) altered the subunit composition of NMDAR complexes with subsequent effects on calcium-sensitive signaling pathways involved in CREB phosphorylation.

Animals↗
PubMed1997

Differential expression of the AP-1/CRE-binding proteins FOS and CREB in preproenkephalin mRNA-expressing neurons of the rat parabrachial nucleus after nociceptive stimulation.

Several subgroups in the brainstem parabrachial nucleus (PB), which is a major target for nociresponsive neurons in the medullary and spinal dorsal horn, contain large numbers of preproenkephalin (ppENK) mRNA-expressing neurons. To elucidate how noxious stimuli may regulate ppENK transcription in these neurons, we have in the present study investigated whether immunoreactivity for the transcription factors FOS and phosphorylated CREB (pCREB), respectively, is displayed in the ppENK mRNA-expressing neurons after peripheral nociceptive stimulation. Rats received injection of formalin into one hindpaw, and were killed 30-80 min later. With a combination of immunohistochemistry and in situ hybridization, we found that only a small number of ppENK mRNA-expressing neurons in PB displayed FOS-immunoreactivity after nociceptive stimulation. In contrast, large numbers of ppENK mRNA-expressing neurons displayed pCREB-like immunoreactivity after nociceptive stimulation. Most of the ppENK mRNA/pCREB-expressing neurons were found in the Kölliker-Fuse and internal lateral subnuclei, but many double-labeled cells were also seen in the ventral lateral and central lateral subnuclei. In addition, a cluster of ppENK mRNA/pCREB-expressing neurons was found in the medial part of the medial parabrachial nucleus. Our findings suggest that CREB rather than FOS regulates nociceptive-related second messenger activation of ppENK transcription in parabrachial neurons.

Animals↗
PubMed2000

Learning-associated activation of nuclear MAPK, CREB and Elk-1, along with Fos production, in the rat hippocampus after a one-trial avoidance learning: abolition by NMDA receptor blockade.

It is widely accepted that the formation of long-term memory (LTM) requires neuronal gene expression, protein synthesis and the remodeling of synaptic contacts. From mollusk to mammals, the cAMP/PKA/CREB signaling pathway has been shown to play a pivotal role in the establishment of LTM. More recently, the MAPK cascade has been also involved in memory processing. Here, we provide evidence for the participation of hippocampal PKA/CREB and MAPK/Elk-1 pathways, via activation of NMDA receptors, in memory formation of a one-trial avoidance learning in rats. Learning of this task is associated with an activation of p44 and p42 MAPKs, CREB and Elk-1, along with an increase in the levels of the catalytic subunit of PKA and Fos protein in nuclear-enriched hippocampal fractions. These changes were blocked by the immediate posttraining intra-hippocampal infusion of APV, a selective blocker of glutamate NMDA receptors, which renders the animals amnesic for this task. Moreover, no changes were found in control-shocked animals. Thus, inhibitory avoidance training in the rat is associated with an increase in the protein product of an IEG, c-fos, which occurs concomitantly with the activation of nuclear MAPK, CREB and Elk-1. NMDA receptors appear to be a necessary upstream step for the activation of these intracellular cascades during learning.

2-Amino-5-phosphonovalerate↗
PubMed1998

Cyclic AMP potentiates growth hormone-dependent differentiation of 3T3-F442A preadipocytes: possible involvement of the transcription factor CREB.

We have examined the effects of cyclic AMP on the differentiation of 3T3-F442A preadipocytes. High concentrations of intracellular cyclic AMP potently inhibited differentiation whereas low concentrations of intracellular cyclic AMP, induced by a number of different agents, promoted differentiation. To analyse these effects of cyclic AMP more closely, we developed a two-phase protocol for the differentiation of 3T3-F442A cells. Growth hormone (GH) was necessary to prime confluent cells during the first phase, following which, the addition of insulin and other adipogenic agents then promoted terminal differentiation. Cyclic AMP potentiated the priming action of GH but exerted an inhibitory effect on terminal differentiation when added to cells which had previously been primed with GH showing that the effects of cyclic AMP on preadipocyte differentiation are stage-dependent. We analysed the stimulatory effects of cyclic AMP during GH priming and found that cyclic AMP induced phosphorylation of the cyclic AMP response element (CRE) binding protein CREB and activated transcription of a CRE-linked reporter gene. Furthermore, GH also stimulated CREB phosphorylation and activation and this effect was potentiated by cyclic AMP. These results suggest a mechanism for the synergistic priming of preadipocytes for terminal differentiation by cyclic AMP and GH via the activation of differentiation genes containing CREs.

1-Methyl-3-isobutylxanthine↗
PubMed2001

Alterations of AP-1 and CREB protein DNA binding in rat supraoptic and paraventricular nuclei by acute and repeated hyperosmotic stress.

Electrophoretic mobility shift assays were used to analyze Fos and CREB protein-DNA-interactions in the rat hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. After intraperitoneal administration of normal saline, PVN (but not SON) extracts exhibited a significant 183% increase in binding to the activational protein-1 (AP-1) canonical DNA binding sequence. Hypertonic saline treatment resulted in a approximately 2.5-fold increase in binding by tissue samples from both regions. AP-1 binding by SON extracts after two hypertonic saline injections caused a 307% increase in binding that was significantly greater than binding by PVN extracts (207%). Fos binding was equal in the SON after one and two hypertonic saline injections, but the PVN exhibited less of an increase after two injections. Binding to the canonical cyclic adenosine monophosphate regulatory element (CRE), and phosphorylated CREB (pCREB) supershift binding, indicated pCREB is constitutively expressed. Any experimental treatment (handling and an injection) caused an elevation in binding in the PVN. AP-1 protein complex DNA binding was increased after osmotic stimulation, and SON and PVN exhibit differences in AP-1 DNA binding kinetics, after repeated hypertonic saline stress. Changes in PVN tissue samples were subtle, and may reflect the fact that magnocellular and parvocellular neurons mediate, respectively, fluid homeostasis and stress responses.

Acute Disease↗
PubMed2002

Ultraviolet B (UVB) induction of the c-fos promoter is mediated by phospho-cAMP response element binding protein (CREB) binding to CRE and c-fos activator protein 1 site (FAP1) cis elements.

The ultraviolet B (UVB) portion (280-320 nm) of the ultraviolet spectrum has been shown to contribute to the development of non-melanoma skin cancer in humans. Research in the human keratinocyte cell line, HaCaT, revealed that UVB irradiation caused the upregulation of the transcription factor activator protein-1 (AP-1). The AP-1 complex formed in UVB-irradiated HaCaT cells is specifically composed of c-fos and Jun D. c-Fos expression was induced in a manner that correlated with the UVB-induced activation of AP-1. To investigate how c-fos expression is regulated by UVB irradiation, the role of each of four cis elements within the c-fos promoter was evaluated. Clustered point mutations at the sis inducible element (SIE), serum response element (SRE), c-fos AP-1 site (FAP1), or cyclic AMP response elements (CRE) significantly inhibited UVB induction of the c-fos promoter. This indicated that all four cis elements are required for maximum promoter activity. The CRE and FAP1 elements were the two most active cis elements that mediate the UVB transactivation of c-fos. Homodimers of phosphorylated cAMP response element binding protein (CREB) were induced by UVB irradiation to bind to each of these elements. Therefore, CREB may function as an important regulatory protein in the UVB-induced expression of c-fos.

Binding Sites↗
PubMed2003

An interplay of Sp1, GKLF and CREB-2 controls human Pre-alpha-Inhibitor gene (ITIH3) transcription.

Pre-alpha-Inhibitor is a plasma protease inhibitor and a heterodimeric molecule whose one polypeptide chain is encoded by the ITIH3 gene. In order to understand the expression of this protein that is regulated in health and disease, we have analyzed the 5' flanking region of ITIH3, specifically focussing on its proximal promoter. A combination of methods including wild-type (wt) or mutant promoter linked to a reporter cat gene, co-transfections of cat constructs with expression plasmids for nuclear factors and electrophoretic mobility shift assays revealed that two antagonistic sets of regulatory elements and nuclear proteins are critical for the activity of this promoter. Indeed, several overlapping Sp1/Sp3-binding sites are required for a sustained activity. However, a tripartite complex including CREB-2 and two molecules of the gut-enriched, Krüppel-like factor cooperate to bind to an upstream area whose 3' end overlaps the Sp1-binding sites. The resulting competition between this tripartite complex and Sp1 results in impaired occupancy of Sp1-binding sites by Sp1 and a consequent reduction in ITIH3 transcription. Competition between Sp1 and a Krüppel-like factor for GC-rich sites has been previously reported, but this is the first description of an elaborate tripartite cooperation of two Krüppel-like factors and CREB as a key step in such a competition.

Activating Transcription Factor 4↗
PubMed1997

ATF1 and CREB trans-activate a cell cycle regulated histone H4 gene at a distal nuclear matrix associated promoter element.

Proteins of the ATF/CREB class of transcription factors stimulate gene expression of several cell growth-related genes through protein kinase A-related cAMP response elements. The promoter activity of cell cycle regulated histone H4 genes is regulated by at least four principal cis-acting elements which mediate G1/S phase control and/or enhancement of transcription during the cell cycle. Using protein-DNA interaction assays we show that the H4 promoter contains two ATF/CREB recognition motifs which interact with CREB, ATF1, and ATF2 but not with ATF4/CREB2. One ATF/CRE motif is located in the distal promoter at the nuclear matrix-associated Site IV, and the second motif is present in the proximal promoter at Site I. Both ATF/CRE motifs overlap binding sequences for the multifunctional YY1 transcription factor, which has previously been shown to be nuclear matrix associated. Subnuclear fractionation reveals that there are two ATF1 isoforms which appear to differ with respect to DNA binding activity and partition selectively between nuclear matrix and nonmatrix compartments, consistent with the role of the nuclear matrix in regulating gene expression. Site-directed mutational studies demonstrate that Site I and Site IV together support ATF1- and CREB-induced trans-activation of the H4 promoter. Thus, our data establish that ATF/CREB factors functionally modulate histone H4 gene transcription at distal and proximal promoter elements.

Activating Transcription Factor 1↗
PubMed1988

Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB.

A nuclear protein, CREB, has been isolated from rat brain and shown to stimulate transcription of the cyclic AMP-responsive gene somatostatin as a dimer. Biochemical analysis suggests that dimerization and transcriptional efficacy of CREB protein in vitro are regulated by phosphorylation. These findings demonstrate that cellular signals can modulate gene expression by regulating the covalent modification of pre-existing nuclear factors.

Acetyltransferases↗
PubMed2001

Transcriptional regulation by the phosphorylation-dependent factor CREB.

The transcription factor CREB -- for 'cyclic AMP response element-binding protein' -- functions in glucose homeostasis, growth-factor-dependent cell survival, and has been implicated in learning and memory. CREB is phosphorylated in response to various signals, but how is specificity achieved in these signalling pathways?

Activating Transcription Factor 1↗
PubMed2006

A novel MASH1 enhancer with N-myc and CREB-binding sites is active in neuroblastoma.

Neuroblastoma is one of the most common solid tumors in childhood. With the aim of developing a targeting vector for neuroblastoma, we cloned and characterized an enhancer in the 5'-flanking regions of the MASH1 gene by a random-trap method from a 36 kb cosmid DNA. The enhancer-containing clone was identified by the expression of GFP when transfected into neuroblastoma cell lines. The enhancer-luciferase activity is higher in neuroblastoma cell lines, IMR32, BE2 and SH-SY5Y, compared with those in non-neuroblastoma cell lines, U1242 glioma, N417 small cell lung cancer and EOMA hemangioma. The core enhancer was determined within a 0.2 kb fragment, yielding three- to fourfold higher activity than that of the MASH1 promoter alone in IMR32 and BE2. This area possesses GATA- and CREB-binding sites, as well as the E-box. EMSA on this area demonstrated that CREB/ATF could bind the DNA. Chromatin immunoprecipitation assay revealed that N-myc, CREB, and co-activators CBP and PCAF, but not HDAC1, are bound to the core enhancer at the same time as the co-activators and N-myc bind to the promoter. This supports the idea that the commonly overexpressed genes HASH1 and N-myc are regulated in concert, confirming their importance as prognostic markers or targets for therapy.

Animals↗
PubMed2004

Repeated quinpirole treatment increases cAMP-dependent protein kinase activity and CREB phosphorylation in nucleus accumbens and reverses quinpirole-induced sensorimotor gating deficits in rats.

Sensorimotor gating, which is severely disrupted in schizophrenic patients, can be measured by assessing prepulse inhibition of the acoustic startle response (PPI). Acute administration of D2-like receptor agonists such as quinpirole reduces PPI, but tolerance occurs upon repeated administration. In the present study, PPI in rats was reduced by acute quinpirole (0.1 mg/kg, s.c.), but not following repeated quinpirole treatment once daily for 28 days. Repeated quinpirole treatment did not alter the levels of basal-, forskolin- (5 microM), or SKF 82958- (10 microM) stimulated adenylate cyclase activity in the nucleus accumbens (NAc), but significantly increased cAMP-dependent protein kinase (PKA) activity. Phosphorylation of cAMP response element-binding protein (CREB) was significantly greater in the NAc after repeated quinpirole treatment than after repeated saline treatment with or without acute quinpirole challenge. Activation of PKA by intra-accumbens infusion of the cAMP analog, Sp-cAMPS, prevented acute quinpirole-induced PPI disruption, similar to the behavioral effect observed following repeated quinpirole treatment. Thus, repeated quinpirole treatment increases NAc PKA activity and CREB phosphorylation, and this neuroadaptive response might facilitate the recovery of sensorimotor gating in schizophrenia.

Acoustic Stimulation↗
PubMed1999

Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors.

Tumour suppressor genes and growth regulatory genes are frequent targets for methylation defects that can result in aberrant expression and mutagenesis. We have established a methylation map of the promoter region of the neurofibromatosis (NF1) gene and demonstrated functional sensitivity for methylation at specific sites for the SP1 and CRE binding (CREB) proteins in the NF1 regulatory region. We evaluated the methylation status of CpG dinucleotides within five promoter subregions in the human and mouse homologues of the neurofibromatosis (NF1) genes. Three 5' subregions were found to be consistently methylated in all the tissues analysed. In contrast, DNA methylation was absent in the vicinity of the transcription start site bounded by SP1 recognition sequences. Gelshift assays showed that methylation specifically inhibits the CREB transcription factor from binding to its recognition site at the NF1 transcription start site. Furthermore, SP1 elements within the NF1 promoter are methylation sensitive, particularly when methylation is present on the antisense strand. We propose that for NF1 as with several other tumour suppressor genes, CpG methylation occurs in a complex, site-specific manner with the maintenance of a methylation-free promoter region bounded by SP1 binding sites that allow an accessible promoter to be retained. When these SP1 boundaries are breached, methylation can sweep in, rendering the promoter inaccessible for specific methylation-sensitive transcription factors and leading to a loss of functional integrity of the methylation-free CpG island.

Animals↗
PubMed2006

Mitogen- and stress-activated protein kinase 1 is critical for interleukin-1-induced, CREB-mediated, c-fos gene expression in keratinocytes.

c-fos, which encodes a transcription factor of the AP-1 family, is a prototypical immediate-early gene induced by a number of proinflammatory cytokines including interleukin-1 (IL-1), the latter being an important regulator of skin homeostasis. Using the human keratinocyte cell line HaCaT as an in vitro model, we dissected the molecular pathways leading to IL-1-induced c-fos gene induction. Phosphorylation of the transcription factor cAMP response element binding protein (CREB) at Ser133 was found to be essential for IL-1-induced c-fos gene induction and was closely paralleled by protein kinase A (PKA) activation. In contrast to other cell types, the cyclooxygenase/prostaglandin pathway, known to activate the cAMP/PKA cascade, plays little, if any, role in c-fos expression downstream of the IL-1 receptor in keratinocytes. Simultaneous activation of several of the mitogen-activated protein kinase (MAPK) cascades occurred in response to IL-1, but each differentially contributed to c-fos induction by IL-1, with the p38/MAPK being the most crucial of all, the extracellular signal-regulated kinase pathway contributing in an additive manner and the Jun N-terminal kinase pathway playing little, if any, role. We also demonstrate that p38-dependent activation of mitogen- and stress-activated kinase 1 (MSK1), a CREB kinase, is a key step for c-fos gene activation by IL-1. Finally, we identify MSK1 as playing a positive role in the control of cell proliferation of both HaCaT keratinocytes and the A431 human epidermoid carcinoma line.

Animals↗
PubMed2006

Single-stranded RNA viruses inactivate the transcriptional activity of p53 but induce NOXA-dependent apoptosis via post-translational modifications of IRF-1, IRF-3 and CREB.

To characterize the mechanisms underlying apoptosis induced by viral infection, transcriptional activation of genes encoding members of the 'BH3-only' family of proteins was analysed during the course of virus infection. Among these genes, only NOXA is transcriptionally activated by vesicular stomatitis virus (VSV), sendai virus (SV), measles virus, herpes simplex virus, or dsRNA and required for efficient apoptosis of cells. Transcriptional activation of NOXA by VSV or SV is independent of p53, but requires the presence of interferon regulatory factor 1 (IRF-1), IRF-3 and cAMP-responsive element binding protein (CREB). Binding to and transactivation of the NOXA promoter by each of these transcription factors is governed by post-translational modification involving different pathways for each factor. Thus, SV infection activates IRF-3 and CREB by phosphorylation triggered by Toll like receptor 3 signalling, and a pathway involving calcium-independent phopholipase A2, respectively. In addition transactivation induced by IRF-1 during viral infection correlates with a 10 kDa increase in its molecular weight, suggesting a covalent linkage with a previously unknown regulatory polypeptide.

Apoptosis↗
PubMed2000

Action of rolipram on specific PDE4 cAMP phosphodiesterase isoforms and on the phosphorylation of cAMP-response-element-binding protein (CREB) and p38 mitogen-activated protein (MAP) kinase in U937 monocytic cells.

U937 monocytic cells are shown here to express a range of PDE4, cAMP-specific phosphodiesterase (PDE) isoenzymes: the long isoenzymes, PDE4A4, PDE4D5 and PDE4D3, plus the short isoenzyme, PDE4B2. These isoenzymes provide around 76% of the total cAMP PDE activity of U937 cells. The specific activities of the total PDE4A, PDE4B and PDE4D activities were 0.63+/-0.09, 8.8+/-0.2 and 34.4+/-2.9 pmol/min per mg of protein respectively. The PDE4 selective inhibitor, rolipram, inhibited immunopurified PDE4B and PDE4D activities similarly, with IC(50) values of approx. 130 nM and 240 nM respectively. In contrast, rolipram inhibited immunopurified PDE4A activity with a dramatically lower IC(50) value of around 3 nM. Rolipram increased phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion, which implied the presence of both high affinity (IC(50) value approx. 1 nM) and low affinity (IC(50) value approx. 120 nM) components. Rolipram dose-dependently inhibited the interferon-gamma (IFN-gamma)-stimulated phosphorylation of p38 mitogen-activated protein (MAP) kinase in a simple monotonic fashion with an IC(50) value of approx. 290 nM. On this basis, it is suggested that rolipram inhibition of PDE4A4 is involved in regulating CREB phosphorylation but not IFN-gamma-stimulated p38 MAP kinase phosphorylation. PDE4A4 was also selectively activated by challenge of U937 cells with either bacterial lipopolysaccharide (LPS) or IFN-gamma through a process which was attenuated by both wortmannin and rapamycin. It is proposed that the PDE4A4 isoform is involved in compartmentalized cAMP signalling responses in U937 monocytes.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Transcriptional induction of the human renin gene by cyclic AMP requires cyclic AMP response element-binding protein (CREB) and a factor binding a pituitary-specific trans-acting factor (Pit-1) motif.

To delineate the cis-acting elements of the proximal promoter responsible for cyclic AMP (cAMP)-induced human renin gene transcription, 5'-flanking regions of the human renin gene were fused to a luciferase reporter gene and transfected in chorionic cells. Forskolin treatment induced the expression of luciferase by 2.4-fold when the reporter plasmid contained the promoter region (-582 to + 16). Mutation or deletion of the cAMP response element (CRE) diminished (1.7-fold) but did not abolish cAMP-induced transcription, demonstrating that the (-582 to -145) region containing the CRE and the region (-145 to -38) containing a Pit-1 (pituitary-specific trans-acting factor) site were both necessary for cAMP maximal induction. To study the molecular events mediating the cAMP induction, DNase I footprinting and electromobility shift assays (EMSAs) were performed with renin-producing chorionic cell and kidney cortex cell nuclear extracts, showing that the CRE-binding protein (CREB) interacts with the CRE and that tissue-specific factors, distinct from Pit-1, specifically bind the renin Pit-1 motif. Taken together, these results demonstrate that the cAMP response of the human renin gene may involve CREB binding the CRE and tissue-specific factors, different from Pit-1, that interact with the Pit-1 response DNA elements.

Base Sequence↗
PubMed2002

Phosphatidylinositol 3-kinase is a central mediator of NMDA receptor signalling to MAP kinase (Erk1/2), Akt/PKB and CREB in striatal neurones.

Ca2+ influx through NMDA receptors can initiate molecular changes in neurones which may underlie synaptic plasticity, neuronal development, survival and excitotoxicity. Signalling through the MAP kinase (Erk1/2) cascade may be central to these processes. We previously demonstrated that Ca2+-permeable AMPA receptors activate Erkl/2 through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent mechanism. We now report that NMDA receptor activation of Erk1/2 was also blocked by inhibitors of PI 3-kinase (LY 294002, wortmannin). In addition, pre-treatment of neurones with pertussis toxin inhibited NMDA-induced Erk1/2 activation, indicating a role for heterotrimeric Gi/o proteins. PI 3-kinase directs activation of the serine-threonine kinase Akt (PKB). Treatment of striatal neurones with glutamate induced a rapid Ca2+-dependent and PI 3-kinase-dependent phosphorylation of Akt (Ser473), which was not blocked by the Mek inhibitors PD98059 or U0126. Targets for Erk1/2 and Akt pathways include transcription factors. Glutamate-induced phosphorylation of cAMP response element binding protein (CREB; Ser133) was partially blocked with either PD98059, U0126, LY294002 or wortmannin but was very strongly inhibited on co-application of LY294002 and PD98059. We propose that NMDA receptor stimulation can activate Erk1/2 and Akt signalling pathways in a PI 3-kinase dependent manner which may target CREB in the nucleus.

Animals↗