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PubMed2000

Coupling of Ca(2+) to CREB activation and gene expression in intact cerebral arteries from mouse : roles of ryanodine receptors and voltage-dependent Ca(2+) channels.

Pathological changes of the vasculature are characterized by changes in Ca(2+) handling and alterations in gene expression. In neurons and other cell types, [Ca(2+)](i) often drives changes in gene expression. However, the relationship between Ca(2+) signaling and gene expression in vascular smooth muscle is not well understood. This study examines the ability of Ca(2+) influx through voltage-dependent, L-type Ca(2+) channels (VDCCs) and Ca(2+) release through ryanodine receptors (RyRs) to activate the transcription factor, cAMP-responsive element binding protein (CREB), and increase c-fos levels in intact cerebral arteries. Membrane depolarization increased the fraction of nuclei staining for phosphorylated CREB (P-CREB) and levels of c-fos mRNA in intact mouse cerebral arteries. Ryanodine, which inhibits RyRs, increased P-CREB staining and c-fos levels. Forskolin, an activator of adenylyl cyclase, and sodium nitroprusside, an NO donor, increased P-CREB and c-fos levels. Nisoldipine, an inhibitor of VDCCs, reversed the effects of depolarization and ryanodine on P-CREB and c-fos levels, but not the effects of forskolin or sodium nitroprusside. Inhibition of Ca(2+)/calmodulin-dependent protein kinase (CaM kinase) blocked increases in P-CREB and c-fos levels seen with membrane depolarization, suggesting that CaM kinase has an important role in the pathway leading from Ca(2+) influx to CREB-mediated changes in c-fos levels. Our data suggest that membrane depolarization increases [Ca(2+)](i) through activation of VDCCs, leading to increased P-CREB and c-fos, and that RyRs have a profound effect on this pathway by indirectly regulating Ca(2+) entry through VDCCs. These results provide the first evidence of Ca(2+) regulation of CREB and c-fos in arterial smooth muscle.

Animals↗
PubMed2006

Ethanol sensitivity: a central role for CREB transcription regulation in the cerebellum.

BACKGROUND: Lowered sensitivity to the effects of ethanol increases the risk of developing alcoholism. Inbred mouse strains have been useful for the study of the genetic basis of various drug addiction-related phenotypes. Inbred Long-Sleep (ILS) and Inbred Short-Sleep (ISS) mice differentially express a number of genes thought to be implicated in sensitivity to the effects of ethanol. Concomitantly, there is evidence for a mediating role of cAMP/PKA/CREB signalling in aspects of alcoholism modelled in animals. In this report, the extent to which CREB signalling impacts the differential expression of genes in ILS and ISS mouse cerebella is examined. RESULTS: A training dataset for Machine Learning (ML) and Exploratory Data Analyses (EDA) was generated from promoter region sequences of a set of genes known to be targets of CREB transcription regulation and a set of genes whose transcription regulations are potentially CREB-independent. For each promoter sequence, a vector of size 132, with elements characterizing nucleotide composition features was generated. Genes whose expressions have been previously determined to be increased in ILS or ISS cerebella were identified, and their CREB regulation status predicted using the ML scheme C4.5. The C4.5 learning scheme was used because, of four ML schemes evaluated, it had the lowest predicted error rate. On an independent evaluation set of 21 genes of known CREB regulation status, C4.5 correctly classified 81% of instances with F-measures of 0.87 and 0.67 respectively for the CREB-regulated and CREB-independent classes. Additionally, six out of eight genes previously determined by two independent microarray platforms to be up-regulated in the ILS or ISS cerebellum were predicted by C4.5 to be transcriptionally regulated by CREB. Furthermore, 64% and 52% of a cross-section of other up-regulated cerebellar genes in ILS and ISS mice, respectively, were deemed to be CREB-regulated. CONCLUSION: These observations collectively suggest that ethanol sensitivity, as it relates to the cerebellum, may be associated with CREB transcription activity.

Alcoholism↗
PubMed1998

Cyclical alternative exon splicing of transcription factor cyclic adenosine monophosphate response element-binding protein (CREB) messenger ribonucleic acid during rat spermatogenesis.

During spermatogenesis, the levels of cAMP in seminiferous tubules undergo stage-dependent cyclical fluctuations. We show that changes in cAMP levels are accompanied by alternative exon splicing of the RNA encoding the cAMP-responsive transcription factor CREB (cAMP response element-binding protein), expressed in both the Sertoli and germ cells. Exons Y and W are expressed exclusively in the testis, and they introduce stop codons into the normal protein coding frame of CREB. The splicing in of W was shown earlier to activate the internal translation of two alternative products of the CREB messenger RNA (mRNA) containing the DNA-binding domain (I-CREBs). The I-CREBs act as potent inhibitors of activator isoforms of CREB. The functions of the alternatively spliced exon Y are unknown. To investigate whether the splicing of exons W and Y is regulated during spermatogenesis, seminiferous tubules, isolated from adult rats, were dissected into segments representing different stages of the spermatogenic cycle and were analyzed by RT-PCR. The analyses of pooled-tubule segments revealed stage-dependent splicing of both exons W and Y in the CREB transcripts. Single tubules were dissected into smaller segments for greater staging accuracy and were analyzed by RT-PCR for CREB mRNAs containing either exons W or Y, as well as for FSH receptor mRNA. This analysis confirmed that a marked, cycle-dependent variation in CREB mRNA levels was occurring. Maximal splicing of exons W and Y occurs independently at different stages of the spermatogenic cycle, stages II-VI and IX, respectively. The distinct spermatogenic cycle-dependent regulation of the splicing of exons W and Y provides further evidence in support of a functional relevance for CREB-W and Y mRNA isoforms in spermatogenesis.

Alternative Splicing↗
PubMed1999

A dominant role for the Raf-MEK pathway in forskolin, 12-O-tetradecanoyl-phorbol acetate, and platelet-derived growth factor-induced CREB (cAMP-responsive element-binding protein) activation, uncoupled from serine 133 phosphorylation in NIH 3T3 cells.

In this study we describe that platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-acetate (TPA), and forskolin induced CREB (cAMP-responsive element-binding protein) Ser-133 phosphorylation with comparable magnitude and kinetics in NIH 3T3 cells. While forskolin was the most potent activator of CREB, TPA or PDGF modestly increased CREB activity. The role of protein kinase C, protein kinase A, and the Raf-MEK kinase pathway in the activation and Ser-133 phosphorylation of CREB by these three stimuli was investigated. We found that inhibition of the Raf-MEK kinase pathway efficiently blocks transcriptional activation of CREB by all three stimuli. This dominant involvement of Raf-MEK in CREB transcriptional activation seems to be uncoupled from CREB Ser-133 phosphorylation. We further demonstrate that although inhibition of Raf-MEK represses forskolin-induced CREB activation, forskolin by itself failed to activate ERK1/2 and Elk-1 mediated transcription. These results suggest that a basal level of Raf-MEK activity is necessary for both PDGF- and forskolin-induced CREB activation, independent of CREB Ser-133 phosphorylation.

3T3 Cells↗
PubMed2001

The CREB family of activators is required for endochondral bone development.

We have evaluated the importance of the CREB family of transcriptional activators for endochondral bone formation by expressing a potent dominant negative CREB inhibitor (A-CREB) in growth plate chondrocytes of transgenic mice. A-CREB transgenic mice exhibited short-limbed dwarfism and died minutes after birth, apparently due to respiratory failure from a diminished rib cage circumference. Consistent with the robust Ser133 phosphorylation and, hence, activation of CREB in chondrocytes within the proliferative zone of wild-type cartilage during development, chondrocytes in A-CREB mutant cartilage exhibited a profound decrease in proliferative index and a delay in hypertrophy. Correspondingly, the expression of certain signaling molecules in cartilage, most notably the Indian hedgehog (Ihh) receptor patched (Ptch), was lower in A-CREB expressing versus wild-type chondrocytes. CREB appears to promote Ptch expression in proliferating chondrocytes via an Ihh-independent pathway; phospho-CREB levels were comparable in cartilage from Ihh(-/-) and wild-type mice. These results demonstrate the presence of a distinct signaling pathway in developing bone that potentiates Ihh signaling and regulates chondrocyte proliferation, at least in part, via the CREB family of activators.

Animals↗
PubMed2006

cAMP response element-binding (CREB) signalling and ovarian surface epithelial cell survival.

cAMP response-element binding (CREB) transcription factors transduce cell survival responses to peptide hormones and growth factors in normal tissues and mutant CREB proteins are implicated in tumorigenesis. Ovarian cancer most frequently arises from the ovarian surface epithelium (OSE), possibly due to repeat inflammation-associated injury-repair episodes that promote neoplasia. We asked if post-receptor signalling involving the CREB family of proteins plays a role in OSE cell survival. In an ovine ovulation model, abundant expression of phospho-CREB/activating transcription factor (ATF) protein was detected immunohistochemically, strongly localised to OSE cells in the proximity of pre-ovulatory follicles. Treatment of primary sheep OSE cell cultures with LH stimulated cAMP accumulation and reduced apoptosis (caspase 3/7 activity) in response to serum withdrawal. When OSE cells were infected with an adenovirus containing a CRE-luciferase construct, exposure to LH and FSH induced CRE-directed transcription. Finally, when a non-phosphorylatable mutant of CREB (Ad CREB(S133A)) was adenovirally expressed, apoptosis measured by activation of caspases was increased several fold relative to that caused by transfection with wild-type CREB (Ad CREB(WT)) or lacZ (Ad lacZ). To test the potential clinical relevance of these findings, we expressed mutant CREB protein in normal human OSE cells from four women and a series of cell lines derived from human ovarian cancers. Infection with Ad CREB(S133A) markedly increased apoptosis in normal human OSE but had no detectable effect on apoptosis in any of the cancer cell lines. We conclude that CREB/ATF signalling is important for the maintenance of OSE cell survival in vitro and is altered in human cell lines derived from ovarian cancers.

Animals↗
PubMed2001

Effects of chronic ethanol intake and its withdrawal on the expression and phosphorylation of the creb gene transcription factor in rat cortex.

This investigation examined the effects of chronic ethanol treatment (15 days) and its withdrawal (24 h) on the expression and phosphorylation of cyclic AMP-response element-binding (CREB) protein in the rat cortex. The effects of chronic ethanol treatment and withdrawal on protein kinase A (PKA) activity and on the expression of the regulatory RII-beta- and the alpha-subtype catalytic subunits of PKA, and on the protein expression of Ca(2+)/calmodulin-dependent protein kinase IV (CaM kinase IV) and calcineurin in the rat cortex were also investigated. It was found that ethanol withdrawal but not ethanol treatment produced a significant decrease in the phosphorylated CREB (p-CREB) and CaM kinase IV protein levels in the frontal, parietal, and piriform cortex. Ethanol treatment and its withdrawal had no effect on the protein levels of total CREB in the frontal, parietal, and piriform cortex. On the other hand, ethanol treatment produced a significant reduction in the protein levels of CREB, p-CREB, and CaM kinase IV in the cingulate gyrus, and these changes reverted to normal levels during ethanol withdrawal. Total CREB protein levels were significantly higher in the cingulate gyrus during ethanol withdrawal. It was also observed that mRNA levels of CREB were significantly higher in the rat cortex during ethanol withdrawal but not during ethanol treatment. The protein levels of RII-beta- and alpha-subtype catalytic subunits of PKA and PKA activity were not modified in the rat cortex by chronic ethanol treatment and its withdrawal. Furthermore, the expression of calcineurin in the rat cortex was not altered during ethanol treatment and withdrawal. Taken together, these results suggest the possibility that decreased CREB-dependent events in the neurocircuitry of the frontal, parietal, and piriform cortex may play an important role in the phenomenon of alcohol dependence and also that decreased CREB-dependent events in the neurocircuitry of the cingulate gyrus may play a role in alcohol tolerance.

Animals↗
PubMed2003

Phosphorylation of CREB and mechanical hyperalgesia is reversed by blockade of the cAMP pathway in a time-dependent manner after repeated intramuscular acid injections.

Spinal activation of the cAMP pathway produces mechanical hyperalgesia, sensitizes nociceptive spinal neurons, and phosphorylates the transcription factor cAMP-responsive element binding protein (CREB), which initiates gene transcription. This study examined the role of the cAMP pathway in a model of chronic muscle pain by assessing associated behavioral changes and phosphorylation of CREB. Bilateral mechanical hyperalgesia of the paw was induced by administering two injections of acidic saline, 5 d apart, into the gastrocnemius muscle of male Sprague Dawley rats. Interestingly, the increases in immunoreactivity for CREB and phosphorylated CREB (p-CREB) in the spinal dorsal horn occur 24 hr, but not 1 week, after the second injection of acidic saline compared with pH 7.2 intramuscular injections. Spinal blockade of adenylate cyclase prevents the expected increase in p-CREB that occurs after intramuscular acid injection. The reversal of mechanical hyperalgesia by adenylate cyclase or protein kinase A inhibitors spinally follows a similar pattern with reversal at 24 hr, but not 1 week, compared with the vehicle controls. The p-CREB immunoreactivity in the superficial dorsal horn correlates with the mechanical withdrawal threshold such that increases in p-CREB are associated with decreases in threshold. Therefore, activation of the cAMP pathway in the spinal cord phosphorylates CREB and produces mechanical hyperalgesia associated with intramuscular acid injections. The mechanical hyperalgesia and phosphorylation of CREB depend on early activation of the cAMP pathway during the first 24 hr but are independent of the cAMP pathway by 1 week after intramuscular injection of acid.

Acids↗
PubMed2002

Early differential elevation and persistence of phosphorylated cAMP-response element binding protein (p-CREB) in the central nervous system of hens treated with diisopropyl phosphorofluoridate, an OPIDN-causing compound.

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus-ester-induced delayed neurotoxicity in sensitive species. We studied the effect of single dose of DFP on the expression of phosphorylated cAMP-response element binding protein (p-CREB), which is a well known transcription factor involved in several pathways mediating different types of external stimuli. The hens were perfused with neutral buffered formalin at different time points, i.e., 0.5, 1.0, and 2.0 hrs, as well as 1, 2, 5, and 20 days after dosing. The central nervous system regions of the whole brain were dissected and 7-micron sections were stained for either p-CREB immunopositivity or with hematoxylin and eosin. Results indicated an early differential increase of p-CREB immunopositivity in susceptible regions such as cerebellum, brainstem, and midbrain within 2 hrs. These induced levels persisted upto 5 days in these tissues, although the time course of p-CREB immunopositivity was distinctly different for each region. In the cerebellum induction of p-CREB was seen in the granular layer where both the granulocytes and the glial cells showed induction. Increased immunopositivity for p-CREB in the Purkinje cells and in some basket cells of the molecular layer was noticed over time, but the induction was not as great as in the granular layer. Of all the tissues cerebellum showed the strongest intensity of immunopositivity of the cells as well as the highest (absolute) number of pCREB-positive cells. The brainstem showed a similar fluctuating pattern like the cerebellum with the highest percentage increase of the immunoreactive cells at 5 days preceded by the lowest dip in immunopositivity at 2 days. In the midbrain, there was a time-dependent increase in the immunopositivity from 0.5 hr onwards until reaching control levels at 20 days. Immunopositivity was also noted in portions of the spina medularis and spina oblongata. The cerebrum (non-susceptible tissue) of DFP-treated hens did not show much deviation from the controls. The endothelial cells of the susceptible regions showed induction at early time points, in contrast to the absence of induction in cerebrum. Spatial and temporal differences in the immunopositivity pattern indicate probable involvement of CREB-independent pathways also. Overall, the complex induction pattern of p-CREB, along with our earlier observations of the early induction of c-fos, c-jun and Protein Kinase A (PKA) as well as the induction of Calcium2+/Calmodulin dependent Protein Kinase II (CaM kinase II) at later periods, strongly suggest an activator role of CREB mediated pathways that may lead to the clinical development of delayed neurotoxicity.

Animals↗
PubMed2005

The immunosuppressive drugs cyclosporin A and tacrolimus inhibit membrane depolarization-induced CREB transcriptional activity at the coactivator level.

Cyclosporin A and tacrolimus are clinically important immunosuppressive drugs directly targeting the transcription factor nuclear factor of activated T cells (NFAT). Through inhibition of calcineurin phosphatase activity they block the dephosphorylation and thus activation of NFAT. Cyclosporin A and tacrolimus also inhibit other calcineurin-dependent transcription factors including the ubiquitously expressed cAMP response element-binding protein (CREB). Membrane depolarization by phosphorylating CREB on Ser119 leads to the recruitment of its coactivator CREB-binding protein (CBP) that stimulates initiation of transcription. It was unknown at what step in CREB-mediated transcription cyclosporin A and tacrolimus interfere. In transient transfection experiments, using GAL4-CREB fusion proteins and a pancreatic islet beta-cell line, cyclosporin A inhibited depolarization-induced activation of CREB proteins which carried various deletions or mutations throughout their sequence providing no evidence for the existence of a distinct CREB domain conferring cyclosporin A sensitivity. In a mammalian two-hybrid assay, cyclosporin A did not inhibit Ser119-dependent interaction of CREB with its coactivator CBP. Using GAL4-CBP fusion proteins, cyclosporin A inhibited depolarization-induced CBP activity, with cyclosporin A-sensitive domains mapped to both the N- (aa 1-451) and C-terminal (aa 2040-2305) ends of CBP. The depolarization-induced transcriptional activity of the CBP C-terminus was enhanced by overexpression of calcineurin and was inhibited by cyclosporin A and tacrolimus in a concentration-dependent manner with IC50 values (10 and 1 nM, respectively) consistent with their known IC50 values for inhibition of calcineurin. These data suggest that, in contrast to NFAT, cyclosporin A and tacrolimus inhibit CREB transcriptional activity at the coactivator level.

Animals↗
PubMed1997

The CREB-binding protein (CBP) cooperates with the serum response factor for transactivation of the c-fos serum response element.

The serum response element is one of the major promoter elements of the immediate early response to extracellular signals. The serum response element includes two main binding sites for proteins: the Ets box, which binds p62(TCF), and the CArG box, which binds p67(SRF). These two proteins are direct targets for signal transduction pathways; p62(TCF) is a nuclear end point of the Ras/mitogen-activated protein kinase pathway, and p67(SRF) is targeted by the Rho/Rac small G-proteins. The mechanism by which the signal is further transduced from the transcription factors to the basal transcriptional machinery is poorly understood. Recent data have suggested that the cAMP-responsive element-binding protein (CREB)-binding protein, a transcriptional adaptor involved in the transactivation through a wide variety of enhancer elements, participates in p62(TCF) activity. We here show that the CREB-binding protein also cooperates in the process of transactivation by p67(SRF). Cotransfections of expression vectors for the CREB-binding protein increased the expression, in response to serum, of reporters under the control of the c-fos serum response element. Interestingly, the C-terminal moiety of the CREB-binding protein was not necessary to observe this effect. The cooperation did not require the Ets box in the serum response element, and the CArG box was sufficient, indicating that the CREB-binding protein is able to cooperate with p67(SRF) in the absence of an Ets protein. Co-immunoprecipitation experiments using cell extracts showed that p67(SRF) could be retained with antibodies directed against the CREB-binding protein, suggesting that the two proteins form a multimolecular complex in live cells. The physical interaction between p67(SRF) and the CREB-binding protein was further confirmed by two-hybrid assays in mammalian cells. Our results indicate that the CREB-binding protein cooperates with p67(SRF) and, thus, suggest that the serum response element is regulated by a multimolecular complex, which includes the CREB-binding protein, p67(SRF), and p62(TCF), with multiple interactions between the components of the complex.

3T3 Cells↗
PubMed2000

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

Activating Transcription Factor 2↗
PubMed2002

Behavioral analysis of CREB alphadelta mutation on a B6/129 F1 hybrid background.

The cyclic AMP (cAMP)-response element binding protein (CREB) is an activity-dependent transcription factor that plays a role in synaptic plasticity and memory storage in Aplysia, Drosophila, and rodents. Mice with targeted deletions of two CREB isoforms (alpha and delta; CREB alphadelta mice) have been characterized on a mixed genetic background of C57BL/6 (B6) and 129/SvEv (129), as well as on a defined F1 hybrid of B6 and FVB/N, and these results suggest that the phenotype of CREB alphadelta mice depends critically on genetic background. In an examination of the hypothesis that the role of CREB in learning and memory can be influenced by strain differences, we analyzed mice with the CREB alphadelta mutation on an F1 hybrid background of B6 and 129 strains. CREB alphadelta mice on this background had impaired short-term and long-term cued and contextual fear conditioning and normal spatial learning in the Morris water maze. Our results suggest that at least some aspects of hippocampal function are normal in CREB alphadelta mice, and that CREB alphadelta mice on the B6/129 F1 background have alterations in amygdala function. These studies underscore the importance of controlling for genetic background in the behavioral analysis of knockout and transgenic mice.

Animals↗
PubMed2004

Modulation of CREB expression and phosphorylation in the rat nucleus accumbens during nicotine exposure and withdrawal.

The nucleus accumbens region of the brain has been shown to play a role in reward and reinforcing mechanisms of drugs of abuse. To understand the molecular mechanisms of nicotine addiction, the present investigation examined the effects of acute and chronic nicotine treatment and its withdrawal on cAMP-responsive element binding (CREB) protein expression and phosphorylation (serine-133) in nucleus accumbens (NAc) structures of rats. it was found that acute treatment (1 and 18 hr of withdrawal) with nicotine had no effects on total creb and phosphorylated CREB (p-CREB) protein levels in shell or core structures of rat NAc. On the other hand, 18-hr withdrawal after chronic nicotine exposure produced significant reductions in the total CREB and p-CREB protein levels in the shell but not in core structures of nac. interestingly, nicotine withdrawal (1 hr) after chronic exposure maintained normal levels of total CREB and p-CREB protein levels in the shell and core structures of NAc. These results suggest the possibility that decreased CREB activity in the shell of NAc may be associated with abnormal reward mechanisms during nicotine withdrawal after chronic exposure.

Animals↗
PubMed1996

Signaling pathways for antigen receptor-mediated induction of transcription factor CREB in B lymphocytes.

We previously reported that cross-linking surface immunoglobulin (sIg) leads to induction of the transcription factor CREB in B lymphocytes through phosphorylation at Ser133, despite the lack of an increase in cAMP. Further, cAMP-raising agents fail to induce CREB Ser133 phosphorylation and CRE-dependent gene expression in these cells, which differs sharply from the situation in PC12 rat pheochromocytoma cells where CREB responds to elevation of cAMP through the activity of protein kinase A. In this study, we characterized the signal transduction pathways leading from sIg engagement to CREB activation. By using specific inhibitors for protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), and protein kinase A (PKA), we found that anti-Ig-induced CREB Ser133 phosphorylation depends on PKC, but does not require activation of PKA or CaM kinase II. The differential responsiveness of CREB to forskolin in PC12 cells and BAL-17 B cells may relate to the more marked elevation of cAMP in the former as opposed to the latter; however, high concentrations of dbcAMP which should readily enter B cells and artificially increase cAMP levels still failed to induce CREB Ser133 phosphorylation, even in conjunction with a phosphodiesterase inhibitor. Taken together, the cAMP/PKA pathway does not appear to be as active a contributor to CREB phosphorylation in B lymphocytes as in PC12 cells, and does not appear to be involved in sIg-induced, PKC-dependent, CREB activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2000

Persistent CREB phosphorylation with protection of hippocampal CA1 pyramidal neurons following temporary occlusion of the middle cerebral artery in the rat.

Phosphorylation of the DNA-binding transcription factor, cyclic AMP response element binding protein (CREB), was immunohistochemically examined in rat brain hippocampal CA1 in order to examine the ischemic vulnerability of this region from the viewpoint of CREB activation. The rat brain had been subjected to 90-min focal ischemia followed by various periods of recirculation. Focal ischemia was induced by occlusion of the middle cerebral artery using the intraluminal suture method. CA1 pyramidal neurons in the sham animals showed definite immunoreactivity with anti-CREB antibody, which binds to both unphosphorylated and phosphorylated CREB, while reactivity with anti-phosphorylated CREB antibody was barely detectable in these neurons. In contrast, at 3.5 h of recirculation, a significant increase in the number of phosphorylated CREB-positive neurons was noted in the CA1 on both sides, and the increase continued until 48 h of recirculation with a tendency for gradual decline. At each period, the ischemic side showed a more marked increase in the number of immunoreactive cells as compared to the nonischemic side. Cresyl violet staining revealed CA1 pyramidal neurons to be maintained intact until 14 day of recirculation, at which time CREB phosphorylation has returned to the control level. Transient global ischemia is known to induce only mild CREB phosphorylation in the CA1 followed by a frank neuronal loss in this region. These data suggest that CREB phosphorylation can be persistently activated in CA1 neurons after focal ischemia and that this phenomenon may be closely associated with protection of these neurons.

Animals↗
PubMed2002

Activation of HTLV-I transcription in the presence of Tax is independent of the acetylation of CREB-2 (ATF-4).

We have previously demonstrated that the bZIP transcription factor CREB-2, also called ATF-4, trans-activates, in association with the viral protein Tax, the human T-cell leukemia virus type I (HTLV-I) promoter. In this study, we have examined whether CREB-2 acetylation affects transcriptional activation mediated by Tax. We present evidence that CREB-2 is acetylated in vitro and in vivo. CREB-2 is acetylated in two regions: the basic domain of the bZIP (from amino acid residue 270 to 300) and the short basic domain (from 342 to 351) located downstream from the bZIP. We also demonstrate that CREB-2 is acetylated by p300/CBP but not by p/CAF. Moreover, replacement of lysine by arginine in the basic domains decreases the trans-activating capacity of CREB-2. However, in the presence of Tax, the HTLV-I transcription remains fully activated by these CREB-2 mutants. Although we cannot totally exclude that the mutations could also affect CREB-2 structure and activity independent of acetylation, our results suggest that activation of the viral promoter in the presence of Tax is independent of the CREB-2 acetylation.

Acetylation↗
PubMed2002

Transcription factor CREB and its stimulus-dependent phosphorylation in cell and explant cultures of the bovine subcommissural organ.

The subcommissural organ (SCO) is an ependymal brain gland that synthesizes and secretes glycoproteins. Very little is known about the signal transduction cascades operating in this organ and their impact on gene expression. An important transcription factor that regulates gene expression in glial cells and neurons is the cyclic-AMP-responsive element binding protein (CREB), which is activated by phosphorylation of the serine residue 133. Here, we analyzed the presence of CREB in bovine SCO cells and its phosphorylation by drugs that activate cyclic-AMP-dependent or calcium-dependent signal transduction pathways. We also investigated the effects of three natural signaling molecules, serotonin (5HT), substance P (SP) and ATP, on CREB phosphorylation and on the second messengers cyclic AMP and calcium. Investigations were performed with cell and explant cultures by using immunocytochemistry, immunoblot, enzyme-linked immunosorbent assay, and the Fura-2 technique. A strong immunosignal for total (phosphorylated and unphosphorylated) CREB was found in virtually all SCO cells. Total CREB levels did not change upon stimulation. Phosphorylated (p)CREB levels were low in unstimulated cells and significantly elevated by drugs that increase the levels of cyclic AMP or free calcium ions. pCREB was also induced by SP and ATP; both substances increased the intracellular calcium concentration but did not affect the formation of intracellular cyclic AMP. 5HT did not influence the phosphorylation of CREB, the intracellular calcium concentration, or the formation of cyclic AMP. Our data identify CREB as an SCO transcription factor that can be activated by the second messengers cAMP and calcium. SP and ATP stimulate the phosphorylation of CREB apparently via a calcium-dependent mechanism and are thus involved in the control of gene expression in the bovine SCO.

Animals↗